Compounds, methods and pharmaceutical compositions for treating cellular damage, such as neural or cardiovascular tissue damage

ABSTRACT

This invention relates to compounds, pharmaceutical compositions, and methods of using the disclosed compounds for inhibiting PARP.

FIELD OF THE INVENTION

The present invention relates to inhibitors of the nuclear enzymepoly(adenosine 5′-diphospho-ribose) polymerase [“poly(ADP-ribose)polymerase” or “PARP”, which is also referred to as ADPRT (NAD:protein(ADP-ribosyl transferase (polymersing)), pADPRT (poly(ADP-ribose)transferase) and PARS (poly(ADP-ribose) synthetase) and providescompounds and compositions containing the disclosed compounds. Moreover,the present invention provides methods of using the disclosed PARPinhibitors to prevent and/or treat tissue damage resulting from celldamage or death due to necrosis or apoptosis; neural tissue damageresulting from, for example, ischemia and reperfusion injury, such ascerebral ischemic stroke, head trauma or spinal cord injury;neurological disorders and neurodegenerative diseases, such as, forexample, Parkinson's or Alzheimer's diseases and multiple sclerosis; toprevent or treat vascular stroke; to treat or prevent cardiovasculardisorders, such as, for example, myocardial infarction; to treat otherconditions and/or disorders such as, for example, age-related musculardegeneration, AIDS and other immune senescence diseases, arthritis,atherosclerosis, cachexia, cancer, degenerative diseases of skeletalmuscle involving replicative senescence, diabetes (such as diabetesmellitus), inflammatory bowel disorders (such as colitis and Crohn'sdisease), acute pancreatitis, mucositis, hemorrhagic shock, splanchnicartery occlusion shock, multiple organ failure (such as involving any ofthe kidney, liver, renal, pulmonary, retinal, pancreatic and/or skeltalmuscle systems), acute autoimmune thyroiditis, muscular dystrophy,osteoarthritis, osteoporosis, chronic and acute pain (such asneuropathic pain), renal failure, retinal ischemia, septic shock (suchas endotoxic shock), local and/or remote endothelial cell dysfunction(such are recognized by endo-dependent relaxant responses andup-regulation of adhesion molecules), inflammation and skin aging; toextend the lifespan and proliferative capacity of cells, such as, forexample, as general mediators in the generation of oxidants,proinflammatory mediators and/or cytokines, and general mediators ofleukocyte infiltration, calcium ion overload, phospholipid peroxidaion,impaired nitric oxide metabolism and/or reduced ATP production; to altergene expression of senescent cells; or to radiosensitize hypoxic tumorcells.

BACKGROUND OF THE INVENTION

PARP (EC 2.4.2.30), also known as PARS (for poly(ADP-ribose)synthetase), or ADPRT (for NAD:protein (ADP-ribosyl) transferase(polymerising)), or pADPRT (for poly(ADP-ribose) transferase), is amajor nuclear protein of 116 kDa. It is present in almost alleukaryotes. The enzyme synthesizes poly(ADP-ribose), a branched polymerthat can consist of over 200 ADP-ribose units from NAD. The proteinacceptors of poly(ADP-ribose) are directly or indirectly involved inmaintaining DNA integrity. They include histones, topoisomerases, DNAand RNA polymerases. DNA ligases, and Ca²⁺- and Mg ²⁺-dependentendonucleases. PARP protein is expressed at a high level in manytissues, most notably in the immune system, heart, brain and germ-linecells. Under normal physiological conditions, there is minimal PARPactivity. However, DNA damage causes an immediate activation of PARP byup to 500-fold. Among the many functions attributed to PARP is its majorrole in facilitating DNA repair by ADP-ribosylation and thereforecoordinating a number of DNA repair proteins. As a result of PARPactivation, NAD levels significantly decline. While many endogenous andexogenous agents have been shown to damage DNA and activate PARP,peroxynitrite, formed from a combination of nitric oxide (NO) andsuperoxide. appears to be a main perpetrator responsible for variousreported disease conditions in vivo. e.g., during shock and inflammation

Extensive PARP activation leads to severe depletion of NAD in cellssuffering from massive DNA damage. The short life of poly(ADP-ribose)(half-life <1 min) results in a rapid turnover rate. Oncepoly(ADP-ribose) is formed, it is quickly degraded by the constitutivelyactive poly(ADP-ribose) glycohydrolase (PARG), together withphosphodiesterase and (ADP-ribose) protein lyase. PARP and PARG form acycle that converts a large amount of NAD to ADP-ribose. In less than anhour, over-stimulation of PARP can cause a drop of NAD and ATP to lessthan 20% of the normal level. Such a scenario is especially detrimentalduring ischaemia when deprivation of oxygen has already drasticallycompromised cellular energy output. Subsequent free radical productionduring reperfusion is assumed to be a major cause of tissue damage. Partof the ATP drop, which is typical in many organs during ischaemia andreperfusion, could be linked to NAD depletion due to poly(ADP-ribose)turnover. Thus, PARP or PARG inhibition is expected to preserve thecellular energy level to potentiate the survival of ischaemic tissuesafter insult.

Poly(ADP-ribose) synthesis is also involved in the induced expression ofa number of genes essential for inflammatory response. PARP inhibitorssuppress production of inducible nitric oxide synthase (iNOS) inmacrophages, P-type selectin and intercellular adhesion molecule-1(ICAM-1) in endothelial cells. Such activity underlies the stronganti-inflammation effects exhibited by PARP inhibitors. PARP inhibitionis able to reduce necrosis by preventing translocation and infiltrationof neutrophils to the injured tissues. (Zhang, J. “PARP inhibition: anovel approach to treat ischaemia/reperfusion and inflammation-relatedinjuries”, Chapter 10 in Emerging Drugs (1999) 4: 209-221 AshleyPublications Ltd.. and references cited therein.)

PARP production is activated bv damaged DNA fragments which, onceactivated, catalyzes the attachment of up to 100 ADP-ribose units to avariety of nuclear proteins. including histones and PARP itself. Duringmajor cellular stresses the extensive activation of PARP can rapidlylead to cell damage or death through depletion of energy stores. As fourmolecules of ATP are consumed for every molecule of NAD (the source ofADP-ribose and PARP substrate) regenerated. NAD is depleted by massivePARP activation and, in the efforts to re-synthesize NAD, ATP may alsobe depleted.

It has been reported that PARP activation plays a key role in both NMDA-and NO-induced neurotoxicity. This has been demonstrated in corticalcultures and in hippocampal slices wherein prevention of toxicity isdirectly correlated to PARP inhibition potency (Zhang et al., “NitricOxide Activation of Poly(ADP-Ribose) Synthetase in Neurotoxicity”,Science, 263:687-89 (1994) and Wallis et al., “Neuroprotection AgainstNitric Oxide Injury with Inhibitors of ADP-Ribosylation”, NeuroReport,5:3, 245-48 (1993)). The potential role of PARP inhibitors in treatingneurodegenerative diseases and head trauma has thus been recognized evenif the exact mechanism of action has not yet been elucidated (Endres etal., “Ischemic Brain Injury is Mediated by the Activation ofPoly(ADP-Ribose)Polymerase”. J. Cereb. Blood Flow Metabol., 17:1143-51(1997) and Wallis et al., Traumatic Neuroprotection with Inhibitors ofNitric Oxide and ADP-Ribosylation, Brain Res., 710:169-77 (1996)).

Similarly, it has been demonstrated that single injections of PARPinhibitors have reduced the infarct size caused by ischemia andreperfusion of the heart or skeletal muscle in rabbits. In thesestudies, a single injection 3-amino-benzamide (10 mg/kg), either oneminute before occlusion or one minute before reperfusion, caused similarreductions in infarct size in the heart (32-42%) while1,5-dihydroxyisoquinoline (1 mg/kg), another PARP inhibitor, reducedinfarct size by a comparable degree (38-48%). Thiemermrann et al.,“Inhibition of the Activity of Poly(ADP Ribose) Synthetase ReducesIschemia-Reperfusion Injury in the Heart and Skeletal Muscle”, Proc.Natl. Acad. Sci. USA. 94:679-83 (1997). These results make it reasonableto suspect that PARP inhibitors could salvage previously ischemic heartor skeletal muscle tissue.

PARP activation can also be used as a measure of damage followingneurotoxic insults following over-exposure to any of glutamate (via NMDAreceptor stimulation), reactive oxygen intermediates, amyloid β-protein,N-methyl-4-phenyl-1,2,3.6-tetrahydropyridine (MPTP) or its activemetabolite N-methyl-4-phenylpyridine (MPP⁺), which participate inpathological conditions such as stroke, Alzheimer's disease andParkinson's disease. Zhang et al., “Poly(ADP-Ribose) SynthetaseActivation: An Early Indicator of Neurotoxic DNA Damage”, J. Neurochem.,65:3. 1411-14 (1995). Other studies have continued to explore the roleof PARP activation in cerebellar granule cells in vitro and in NPTPneurotoxicity. Cosi et al., “Poly(ADP-Ribose) Polymerase (PARP)Revisited. A New Role for an Old Enzyme: PARP Involvement inNeurodegeneration and PARP Inhibitors as Possible NeuroprotectiveAgents”, Ann. N. Y. Acad. Sci., 825:366-79 (1997); and Cosi et al.,“Poly(ADP-Ribose) Polymerase Inhibitors Protect Against MPTP-inducedDepletions of Striatal Dopamine and Cortical Noradrenaline in C57B1/6Mice”. Brain Res., 729:264-69 (1996). Excessive neural exposure toglutamate, which serves as the predominate central nervous systemneurotransmitter and acts upon the N-methyl-D-aspartate (NMDA) receptorsand other subtype receptors, most often occurs as a result of stroke orother neurodegenerative processes. Oxygen deprived neurons releaseglutamate in great quantities during ischemic brain insult such asduring a stroke or heart attack. This excess release of glutamate inturn causes over-stimulation (excitotoxicity) of N-methyl-D-aspartate(NMDA), AMPA, Kainate and MGR receptors, which open ion channels andpermit uncontrolled ion flow (e.g., Ca²⁺ and Na⁺ into the cells and K⁺out of the cells) leading to overstimulation of the neurons. Theover-stimulated neurons secrete more glutamate, creating a feedback loopor domino effect which ultimately results in cell damage or death viathe production of proteases, lipases and free radicals. Excessiveactivation of glutamate receptors has been implicated in variousneurological diseases and conditions including epilepsy, stroke,Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis(ALS), Huntington's disease, schizophrenia, chronic pain, ischeina andneuronal loss following hypoxia, hypoglycemia, ischemia, trauma, andnervous insult. Glutamate exposure and stimulation has also beenimplicated as a basis for compulsive disorders, particularly drugdependence. Evidence includes findings in many animal species. as wellas in cerebral cortical cultures treated with glutamate or NMDA. thatglutamate receptor antagonists (i.e., compounds which block glutamatefrom binding to or activating its receptor) block neural damagefollowing vascular stroke. Dawson et al., “Protection of the Brain fromIschemia”, Cerebrovascular Disease. 319-25 (H. Hunt Batjer ed., 1997).Attempts to prevent excitotoxicity by blocking NMDA, AMPA, Kainate andMGR receptors have proven difficult because each receptor has multiplesites to which glutamate may bind and hence finding an effective mix ofantagonists or universal antagonist to prevent binding of glutamate toall of the receptor and allow testing of this theory, has beendifficult. Moreover, many of the compositions that are effective inblocking the receptors are also toxic to animals. As such, there ispresently no known effective treatment for glutamate abnormalities.

The stimulation of NMDA receptors by glutamate. for example, activatesthe enzyme neuronal nitric oxide synthase (nNOS), leading to theformation of nitric oxide (NO), which also mediates neurotoxicity. NMDAneurotoxicity may be prevented by treatment with nitric oxide synthase(NOS) inhibitors or through targeted genetic disruption of nNOS invitro. Dawson et al., “Nitric Oxide Mediates Glutamate Neurotoxicity inPrimary Cortical Cultures”, Proc. Natl. Acad. Sci. USA. 88:6368-71(1991); and Dawson et al., “Mechanisms of Nitric Oxide-mediatedNeurotoxicity in Primary Brain Cultures”, J. Neurosci., 13:6, 2651-61(1993), Dawson et al., “Resistance to Neurotoxicity in Cortical Culturesfrom Neuronal Nitric Oxide Synthase-Deficient Mice”, J. Neurosci., 16:8,2479-87 (1996), ladecola. “Bright and Dark Sides of Nitric Oxide inIschemic Brain Injury”, Trends Neurosci., 20:3, 132-39 (1997), Huang etal.. “Effects of Cerebral Ischemia in Mice Deficient in Neuronal NitricOxide Synthase”, Science. 265:1883-85 (1994), Beckman et al.,“Pathological Implications of Nitric Oxide, Superoxide and PeroxyntriteFormation”, Biochem. Soc. Trans., 21:330-34 (1993), and Szabóet al.,“DNA Strand Breakage, Activation of Poly(ADP-Ribose) Synthetase, andCellular Energy Depletion are Involved in the Cytotoxicity inMacrophages and Smooth Muscle Cells Exposed to Peroxynitrite”, Proc.Natl. Acad. Sci. USA, 93:1753-58 (1996).

It is also known that PARP inhibitors, such as 3-amino benzamide, affectDNA repair generally in response, for example, to hydrogen peroxide orgamma-radiation. Cristovao et al., “Effect of a Poly(ADP-Ribose)Polymerase Inhibitor on DNA Breakage and Cytotoxicity Induced byHydrogen Peroxide and γ-Radiation,” Terato., Carcino., and Muta.,16:219-27 (1996). Specifically, Cristovao et al. observed aPARP-dependent recovery of DNA strand breaks in leukocytes treated withhydrogen peroxide.

PARP inhibitors have been reported to be effective in radiosensitizinghypoxic tumor cells and effective in preventing tumor cells fromrecovering from potentially lethal damage of DNA after radiationtherapy, presumably by their ability to prevent DNA repair. U.S. Pat.Nos. 5,032,617; 5,215,738; and 5,041,653.

Evidence also exists that PARP inhibitors are useful for treatinginflammatory bowel disorders, such as colitis. Salzman et al., “Role ofPeroxvnitrite and Poly(ADP-Ribose)Synthase Actiyation ExperimentalColitis,” Japanese J. Pharm., 75, Supp. I:15 (1997). Specifically,Colitis was induced in rats by intraluminal administration of the haptentrinitrobenzene sulfonic acid in 50% ethanol. Treated rats received3-aminobenzamide, a specific inhibitor of PARP activity. Inhibition ofPARP activity reduced the inflammatory response and restored themorphology and the energetic status of the distal colon. See also,Southan et al., “Spontaneous Rearrangement of Aminoalkylithioureas intoMercaptoalkylguanidines, a Novel Class of Nitric Oxide SynthaseInhibitors with Selectivity Towards the Inducible Isoform”, Br. J.Pharm., 117:619-32 (1996); and Szabó et al., “Mercaptoethylguanidine andGuanidine Inhibitors of Nitric Oxide Synthase React with Peroxynitriteand Protect Against Peroxynitrite-induced Oxidative Damage”, J. Biol.Chem., 272:9030-36 (1997).

Evidence also exists that PARP inhibitors are useful for treatingarthritis. Szabó et al., “Protective Effects of an Inhibitor ofPoly(ADP-Ribose)Synthetase in Collagen-Induced Arthritis,” Japanese J.Pharm., 75, Supp. I:102 (1997); Szabó et al., “DNA Strand Breakage.Activation of Poly(ADP-Ribose)Synthetase, and Cellular Energy Depletionare Involved in the Cytotoxicity in Macrophages and Smooth Muscle CellsExposed to Peroxynitrite,” Proc. Natl. Acad. Sci. USA, 93:1753-58 (March1996); and Bauer et al., “Modification of Growth Related EnzymaticPathways and Apparent Loss of Tumorigenicity of a ras-transformed BovineEndothelial Cell Line by Treatment with 5-Iodo-6-amino-1,2-benzopyrone(INH2BP)”, Intl. J. Oncol., 8:239-52 (1996); and Hughes et al.,“Induction of T Helper Cell Hxporesponsiveness in an Experimental Modelof Autoimmunity by Using Nonmitogenic Anti-CD3 Monoclonal Antibody”. J.Immuno., 153:3319-25 (1994).

Further, PARP inhibitors appear to be useful for treating diabetes.Heller et al., “Inactivation of the Poly(ADP-Ribose)Polymerase GeneAffects Oxygen Radical and Nitric Oxide Toxicity in Islet Cells,” J.Biol. Chem., 270:19, 11176-80 (May 1995). Heller et al. used cells frommice with inactivated PARP genes and found that these mutant cells didnot show NAD⁺ depletion after exposure to DNA-damaging radicals. Themutant cells were also found to be more resistant to the toxicity of NO.

PARP inhibitors have been shown to be useful for treating endotoxicshock or septic shock. Zingarelli et al., “Protective Effects ofNicotinamide Against Nitric Oxide-Mediated Delayed Vascular Failure inEndotoxic Shock: Potential Involvement of PolyADP Ribosyl Synthetase.”Shock, 5:258-64 (1996), suggests that inhibition of the DNA repair cycletriggered by poly(ADP ribose) synthetase has protective effects againstvascular failure in endotoxic shock. Zingarelli et al. found thatnicotinamide protects against delayed, NO-mediated vascular failure inendotoxic shock. Zingarelli et al. also found that the actions ofnicotinamide may be related to inhibition of the NO-mediated activationof the energy-consuming DNA repair cycle, triggered by poly(ADP ribose)synthetase. Cuzzocrea, “Role of Peroxynitrite and Activation ofPoly(ADP-Ribose) Synthetase in the Vascular Failure Induced byZymosan-activated Plasma,” Brit. J. Pharm., 122:493-503 (1997).

PARP inhibitors have been used to treat cancer. Suto et al.,“Dihydroisoquinolinones: The Design and Synthesis of a New Series ofPotent Inhibitors of Poly(ADP-Ribose) Polymerase”, Anticancer Drug Des.,7:107-17 (1991). In addition. Suto et al., U.S. Pat. No. 5,177,075,discusses several isoquinolines used for enhancing the lethal effects ofionizing radiation or chemotherapeutic agents on tumor cells. Weltin etal., “Effect of 6(5H)-Phenanthridinone, an Inhibitor of Poly(ADP-ribose)Polymerase, on Cultured Tumor Cells”, Oncol. Res., 6:9, 399-403 (1994),discusses the inhibition of PARP activity, reduced proliferation oftumor cells, and a marked synergistic effect when tumor cells areco-treated with an alkylating drug.

Still another use for PARP inhibitors is the treatment of peripheralnerve injuries, and the resultant pathological pain syndrome known asneuropathic pain, such as that induced by chronic constriction injury(CCI) of the common sciatic nerve and in which transsynaptic alterationof spinal cord dorsal horn characterized by hyperchromatosis ofcytoplasm and nucleoplasm (so-called “dark” neurons) occurs. Mao et al.,Pain, 72:355-366 (1997).

PARP inhibitors have also been used to extend the lifespan andproliferative capacity of cells including treatment of diseases such asskin aging, Alzheimer's disease, atherosclerosis, osteoarthritis,osteoporosis, muscular dystrophy, degenerative diseases of skeletalmuscle involving replicative senescence, age-related musculardegeneration, immune senescence. AIDS, and other immune senescencediseases; and to alter gene expression of senescent cells. WO 98/27975.

Large numbers of known PARP inhibitors have been described in Banasik etal., “Specific Inhibitors of Poly(ADP-Ribose) Synthetase andMono(ADP-Ribosyl)-Transferase”, J. Biol. Chem., 267:3, 1569-75 (1992),and in Banasik et al., “Inhibitors and Activators of ADP-RibosylationReactions”, Molec. Cell. Biochem., 138: 185-97 (1994). However,effective use of these PARP inhibitors, in the ways discussed above, hasbeen limited by the concurrent production of unwanted side-effects(Milam et al., “Inhibitors of Poly(Adenosine Diphosphate-Ribose)Synthesis: Effect on Other Metabolic Processes”, Science, 223:589-91(1984)).

There continues to be a need for effective and potent PARP inhibitorswhich produce minimal side-effects. The present invention providescompounds, compositions for, and methods of, inhibiting PARP activityfor treating and/or preventing cellular, tissue and/or organ damageresulting from cell damage or death due to, for example, necrosis orapoptosis. The compounds and compositions of the present invention arespecifically useful in ameliorating, treating and/or preventing neuraltissue or cell damage, including that following focal ischemia andreperfusion injury. Generally, inhibition of PARP activity spares thecell from energy loss, preventing irreversible depolarization of theneurons and, thus, provides neuroprotection. While not wishing to bebound by any mechanistic theory, the inhibition of PARP activity by useof the compounds, compositions and methods of the present invention isbelieved to protect cells. tissue and organs by protection against theill-effects of reactive free radicals and nitric oxide. The presentinvention therefore also provides methods of treating and/or preventingcells, tissue and/or organs from reactive free radical and/or nitricoxide induced damage or injury.

SUMMARY OF THE INVENTION

The present invention provides compunds which inhibit poly(ADP-ribose)polymerase (“PARP”), compositions containing these compounds and methodsfor using these PARP inhibitors to treat, prevent and/or ameliorate theeffects of the conditions described herein.

In one embodiment, the present invention provides compounds of FormulaI:

or a pharmaceutically acceptable salt, hydrate, prodrug, or mixturesthereof, wherein:

m is zero or one;

n is zero or one;

p is one or two;

Y is a direct bond,

—O—, —N(R₁₀)—, N, or —C(R₈)_(p)—;

Z is O, or S;

X is NR₁₁, —O—, —S—, CR₁₂R₁₃, a bond, —CR₁₂=CR₁₃—, or

—C(R₁₂R₁₃)C(R₁₄R₁₅)—,

W is selected from —CN, —C(R₉)₂, —(N(R₉)₂) where the R₉ substituents maybe combined to form a heteroaryl or C₃-C₈ cycloalkyl optionallycontaining at least one hetero atom in place of a carbon atom,—P(O)₂—OR₉, —P(O)(OR₉)₂, —S(O)₂—R₉, —S(O)₃R₉, —C(O)—R₉, —C(O)—N(R₉)₂,—S(O)₂NR₉, C₃-C₈ cycloalkyl optionally containing at least oneheteroatom in place of a carbon atom, and heteroaryl;

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₁₂, R₁₃, R₁₄, and R₁₅, when present,are independendently: hydrogen, lower (C₁-C₉ straight or branched chain)alkyl, C₃-C₈ cycloalkyl optionally containing at least one heteroatom inplace of a carbon atom, lower (C₂-C₉ straight or branched chain)alkenyl, C₅-C₇ cycloalkenyl, lower (C₁-C₄) alkoxy, aryl, heteroaryl,aralkyl, heteroaralkyl, alkaryl, alkheteroaryl, hydroxy, amino, nitro,halo, nitroso, sulfo, sulfonic acid, or carboxy;

R₉ is hydrogen, lower (C₁-C₉ straight or branched chain) alkyl, C₃-C₈cycloalkyl optionally containing at least one heteroatom in place of acarbon atom, lower (C₂-C₉ straight or branched chain) alkenyl, aryl,heteroaryl, araikyl, heteroaralkyl, alkaryl, alkheteroaryl, hydroxy,lower (C₁-C₄) alcohol, lower (C₁-C₄) alkoxy, amino, or carboxy; and

R₁₀ and R₁₁, are independently: hydrogen, lower (C₁-C₉ straight orbranched chain) alkyl, lower (C₂-C₉ straight or branched chain) alkenyl,aryl, aralkyl, alkaryl, halo, hydroxy, lower (C₁-C₄) alkoxy, amino, orcarboxy;

each R₁₆ is independently hydrogen or lower (C₁-C₉ straight or branchedchain) alkyl; and

T, when present, is a divalent or trivalent organic residueindependently selected from the group consisting of: lower (C₂-C₉straight or branched chain) alkylene, lower alkenylene, ariylene,aralkylene, and alkarylene;

wherein one, two or three of the hydrogen atoms of said divalent ortrivalent organic residue can be substituted by a moiety selected fromthe group consisting of: lower (C₁-C₉ straight or branched chain) alkyl,cycloalkyl, lower (C₂-C₉ straight or branched chain) alkenyl,cycloalkenyl, aryl, heteroaryl, aralkyl, heteroaryalkyl, alkaryl,alkheteroaryl, halo, trifluoromethyl, hydroxy, lower (C₁-C₄) alkoxy,amino, nitro, trifluoromethyl, alkenyloxy, phenoxy, and benzylox:

wherein one, two, or three carbon atoms in the divalent or trivalentorganic residue can be replaced by a hetero-atom-containing-moietyselected from the group consisting of: phenoxy, phenoxymethyl,phenoxycarbonyl, benzyloxy, —O—,

—S—, —SO₂—, —NR₁SO₂—, —SO₂NR₁—, —NR₁—, and —PO₂—;

wherein any of the lower (C₁-C₉ straight or branched chain) alkyl, C₃-C₈cycloalkyl optionally containing at least one heteroatom in place of acarbon atom, lower (C₂-C₉ straight or branched chain) alkenyl, aryl,heteroaryl, aralkyl, and alkaryl groups can be independently substitutedwith one, two or three substituents selected from the group consistingof (C₁-C₉ lower straight or branched chain) alkyl, C₃-C₈ cycloalkyloptionally containing at least one heteroatom in place of a carbon atom,lower (C₂-C₉ straight or branched chain) alkenyl, cycloalkenyl, aryl,heteroaryl, aralkyl, heteroaralkyl, alkaryl, alkheteroaryl, halo,trifluoromethyl, hydroxy, lower (C₁-C₄) alkoxy, carboxy (such as methoxyor ethoxy), carbonyl, lower alkyl ester (such as methylester orethylester), amino, nitro, trifluoromethyl, alkenyloxy, phenoxy,benzyloxy,

wherein one, two, or three carbon atoms of any of the lower (C₁-C₉straight or branched chain) alkyl, C₃-C₈ cycloalkyl optionallycontaining at least one heteroatom in place of a carbon atom, lower(C₂-C₉ straight or branched chain) alkenyl, aryl, heteroaryl, aralkyl,and alkary groups can be replaced by a hetero-atom-containing-moietyselected from the group consisting of: —O—,

—S—, —SO₂——NR₁SO₂—, —SO₂NR₁—, N, —NR₁—, and —PO₂—.

The dotted line between positions 1 and 2 in chemical formulas hereinwill be recognized to represent a single or double bond.

In another embodiment, the present invention provides compounds ofFormula (II)

or a pharmaceutically acceptable salt, hydrate, prodrug, or mixturesthereof, wherein:

q is zero or one;

Y is N, —CH— or —CH₂;

Z is O;

X is —O—, or a bond;

R₁, R₂, R₃, R₄, R₅, R₆, and R₇, when present, are independently:hydrogen, lower (C₁-C₉ straight or branched chain) alkyl, lower (C₂-C₉straight or branched chain) alkenyl, C₃-C₈ cyloalkyl optionallycontaining at least one heteroatom in place of a carbon atom, (C₅-C₇cycloalkenyl, lower (C₁-C₄) alkoxy, aryl, heteroaryl, aralkyl,heteroaralkyl, alkaryl, alkheteroarly, hydroxy, amino, nitro, halo,nitroso, or carboxy;

R₉ is hydrogen, lower (C₁-C₉ straight or branched chain) alkyl, (C₃-C₈cycloalkyl optionally containing at least one heteroatom in place of acarbon atom, lower (C₂-C₉ straight or branched chain) alkenyl, aryl,heteroaryl, aralkyl, heteroaralkyl, alkaryl, alkheteroaryl, hydroxy,lower (C₁-C₄) alcohol, lower (C₁-C₄) alkoxy, amino, or carboxy; and

T, when present, is a divalent or trivalent organic radicalindependently selected from the group consisting of: lower alkylene,lower alkenylene, C₂-C₄ alkenyloxy, arylene, aralkylene, and alkarylene;

wherein one, two or three of the hydrogen atoms of said divalent ortrivalent organic radical can be substituted by a moiety selected fromthe group consisting of: lower (C₁-C₉ straight or branched chain) alkyl,lower (C₂-C₉ straight or branched chain) alkenyl, anl, aralkyl, alkaryl,halo, trifluoromethyl, hydroxy, lower (C₁-C₄) alkoxy, amino, nitro,trifluoromethyl, alkenyloxy, phenoxy, and benzyloxy;

wherein one, two, or three carbon atoms in the divalent or trivalentorganic radical can be replaced by a hetero-atom-containing-moietyselected from the group consisting of: phenoxy, phenoxycarbonyl,benzyloxy, —O—

—S—, —SO₂—, —NR₁SO₂—, —SO₂NR₁—, —NR₁—, and —PO₂—;

wherein the lower allyl, cyloalkyl optionally containing at least oneheteroatom, lower (C₂-C₉ straight or branched chain) alkenyl, aryl,heteroaryl, aralh,l, heteroaralkyl, alkaryl, and alkheteroaryl groupscan be independently substituted with one, two or three substituentsselected from the group consisting of: lower (C₁-C₉ straight or branchedchain) alkyl, C₃-C₈ cycolalkyl optionally containing at least oneheteratom, lower (C₂-C₉ straight or branched chain) alkenyl, aryl,heteroaryl, aralkyl, heteroaralkyl, alkaryl, alkheteroaryl, halo,trifluoromethyl, hydroxy, lower (C₁-C₄) alkoxy, carboxy (such as methoxyor ethoxy), carbonyl, lower alkyl ester (such as methylester orethylester), amino, nitro, trifluoromethyl, alkenyloxy, phenoxy,benzyloxy,

wherein one, two, or three carbon atoms thereof can be replaced by ahetero-atom-containing-moiety selected from the group consisting of:—O—,

—S—, —SO₂—, —NR₁SO₂—, —SO₂NR₁—, N, —NR₁—, and —PO₂—.

In a further embodiment, the present invention provides compounds ofFormula (III)

wherein

Z and X are oxygen;

q is zero or one;

“alk” is lower alkylene;

R₁₇, R₁₈ and R₁₉ are independently hydrogen or lower alkyl; or

R₁₇ and R₁₈ or R₁₈ and R₁₉ taken together can be a lower alkylene toform a heterocyclic ring; and

R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are independently: hydrogen, lower (C₁-C₉straight or branched chain) alky, C₃-C₈ cycloalkyl optionally containingat least one hetero atom, lower (C₂-C₉ straight or branched chain)alkenyl, lower (C₁-C₄) alkoxy, aryl, heteroaryl, aralkyl, heteroaralkyl,alkaryl, alkheteroaryl, hydroxy, amino, nitro, halo, nitroso, orcarboxy.

In yet a further embodiment, the present invention provides compounds ofFormulas (IV) and (V)

wherein R₁-R₆ are as defined above. Preferably. R₁-R₆ are eachindependently any of hydrogen, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, hydroxyl, amino, nitro, nitroso, carboxy, trifluoromethyl,phenoxy, and benzyloxy.

Preferred embodiments of the present invention include compounds whereinX and Z are oxygen and Y is nitrogen. The preferred forms of thefollowing specific embodiments include, but are not limited to,compounds wherein X and Z are oxwgen and Y is nitrogen.

Preferred embodiments of the present invention include compounds whereineach of W is —CN, m and n are zero and p is one. Of these, furtherpreferred embodiments include compounds where T is —CH₂—, Z and X areoxygen, Y is N and R₁ to R₇ are hydrogen. Preferably, each of T is —CH—,Z and X are oxygen and Y is N

Further preferred embodiments of the present invention include compoundswhere each of W is —(N(R₉)₂), preferably W is —N(R₉)₂ m and n are zeroand p is one. X is preferably oxygen or a bond, Z is oxygen and Y isnitrogen. Of these, preferred embodiments include those compounds whereeach R₉ is independently selected from hydrogen, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, pentyl, isopentyl, methoxy, ethoxy or aminoor where the R₉ substituents combine with the N to form a 5- or6-membered substituted or unsubstituted heterocycloalkyl, optionallysubsituted with an additional oxygen or nitrogen, or combine with the Nto form a 5- or 6-membered substituted or unsubstituted heteroaryl. Thesubstitutions of these preferred embodiments preferably includingmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, methylester,ethylester, benzyl, phenyl, benzoxy, phenoxy, phenoxycarbonyl, one ortwo additional heterocycloalkyls or heteroaryl rings and/or one or twofused benzene rings. Preferred forms of this embodiment includecompounds where T is absent, —CH₂— or —CH₂—CH₂— CH₂S, CH₂SCH₂,CH₂SCH₂CH₂, methoxy or phenoxymethyl. Particularly preferred forms ofthis embodiment include compounds wherein R₉ substituents combine, withthe N of W, to form a

group, which may be optionally substituted by the above-notedsubstitutions, such as, for example, pyridyl, benzyl, phenyl methylester or ethyl ester, and/or fused with 1-2 additional benzene rings.

In a further preferred embodiment of the present invention, each of W is—N(R₉)₂, m is one and n is one, X is oxygen or a bond, Z is oxygen and Yis nitrogen. Of these, prefered embodiments include those compoundewhere each R₉ is independently selected from hydrogen, methyl, ethyl,propyl, isopropyl, butyl, isobutyl, mehanol, ethanol, methoxy, ethoxy oramino or where the R₉ substituents combine with the N to form a 5- or6-membered substituted or unsubstituted heterocycloalkyl, optionallysubsituted with an additional oxygen or nitrogen, or combine with the Nto form a 5- or 6-membered substituted or unsubstituted heteroaryl. Thesubstitutions of these preferred embodiments preferably includingmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, methyl ester, ethylester, benzyl, phenyl, benzoxy, phenoxy, phenoxycarbonyl and pyridyl.Preferred forms of this embodiment include compounds where T is absent,—CH₂— or —CH₂—CH₂—. Particularly preferred forms of this embodimentincludes compounds wherein R₉ substituents form with the N of W to forma

group, which may be optionally substituted by pyridyl, benzyl or phenyland/or fused with 1-2 additional benzene rings. R₁-R₇ are preferablyhydrogen.

Other preferred embodiments of the present invention includes compoundswherein W is either —P(O)₂—OR₉ or —P(O)(OR₉)₂, m is zero and p is one.Of these, each R₉ is preferably independently hydrogen, methyl, ethyl,propyl, isopropyl, butyl or isobutyl. Preferably, in these embodiments,Y is nitrogen, Z is oxygen and X is oxygen. Preferably, m is zero andwhen n is one, R₁₆ is preferably hydrogen. T is preferably —CH₂— or—CH₂CH₂— in these preferred embodiments.

Further preferred embodiments of the present invention include compoundswherein W is either —S(O)₂—R₉, —S(O)₂—OR₉ or —S(O)₂NR₉, m is zero and pis 1. Of these, preferred embodiments include where R₉ is hydrogen, orsubstituted or unsubstituted methyl, ethyl, propyl, isopropyl, butyl orisobutyl, wherein the optional substitution is a 5- or 6-memberedcycloalkyl, optionally subsituted with at least one oxygen or nitrogenor a 5- or 6-membered heteroaryl. Further preferred forms of theseembodiments include compounds where X is oxygen or a bond, Z is oxygen,T is absent or —CH₂—, R₁-R₇ are hydrogen, Y is nitrogen and R₉ ishydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl,benzylcarbonyl, phenyl,

Preferably. R₉ is hydrogen, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, benzyl, or benzylcarbonyl.

Further preferred embodiments of the present invention include compoundswherein W is either —C(O)—R₉ or —C(O)N(R₉)₂, and m is zero Preferably,n=1 when W is C(O)R₉. Of these, preferred embodiments include compoundswhere each R₉ is independently hydrogen, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, hydroxyl, methanol, ethanol,—(CH(OQ))_(p)COOH (where Q is hydrogen, hydroxyl, ethoxy or methoxy), oramino, and T is (—CH₂—)₁₋₄, wherein optionally one of the methenyl unitsis replace with phenoxy, carbonyl or oxygen. Prefered forms of theseembodiments include compounds where X is oxygen or a bond, Z is oxygen,Y is nitrogen and R₁ to R₇ are hydrogen. R₁₆, when present, ispreferably hydrogen.

Further preferred embodiments of the present invention include compoundswherein W is an optionally substituted 5- or 6-membered cycloalkyloptionally containing at least one heteroatom selected from S, O or N,or a heteroaryl, wherein the cycloalkyl or heteraryl may be substitutedor attached to a further 5- or 6-membered cycloalkyl which mayoptionally contain at least one heteroatom, a 5- or 6-membered heterarylor a 5- or 6-membered aryl.

Preferably, the compounds of the invention exhibit an IC₅₀ forinhibiting PARP in vitro, as measured by the methods described herein,of about 20 μM or less, preferably less than about 10 μM, morepreferably less than about 1 μM, most preferably less than about 0.1 μM.

Preferred embodiments of the present invention include the followingcompounds, and neutral forms thereof, where appropriate.,

Broadly, the compounds and compositions of the present invention can beused to treat or prevent cell damage or death due to necrosis orapoptosis, cerebral ischemia and reperfusion injury or neurodegenerativediseases in an animal, such as a human. The compounds and compositionsof the present invention can be used to extend the lifespan andproliferative capacity of cells and thus can be used to treat or preventdiseases associated therewith; they alter gene expression of senescentcells. and they radiosensitize hypoxic tumor cells. Preferably, thecompounds and compositons of the invention can be used to treat orprevent tissue damage resulting from cell damage or death due tonecrosis or apoptosis, and/or effect neuronal activity, either mediatedor not mediated by NMDA toxicity. The compounds of the present inventionare not limited to being useful in treating glutamate mediatedneurotoxicity and/or NO-mediated biological pathways. Further, thecompounds of the invention can be used to treat or prevent other tissuedamage related to PARP activation, as described herein.

The present invention provides compounds which inhibit the in vitroand/or in vivo polymerase activity of poly(ADP-ribose) polymerase(PARP), and compositions containing the disclosed compounds.

The present invention provides methods to inhibit, limit and/or controlthe in vitro and/or in vivo polymerase activity of poly(ADP-ribose)polymerase (PARP) in any of solutions, cells, tissues, organs or organsystems. In one embodiment, the present invention provides methods oflimiting or inhibiting PARP activity in a mammal, such as a human,either locally or systemically.

The present invention provides methods to treat and/or prevent diseases,syndromes and/or conditions exacerbated by or involving the increasedgeneration of PARP. These methods involve application or administrationof the compounds of the present invention to cells, tissues, organs ororgan systems of a person in need of such treatment or prevention.

In one embodiment, the present invention provides methods to treatand/or prevent cardiovascular tissue damage resulting from cardiacischemia or reperfusion injury. Reperfusion injury, for instance, occursat the termination of cardiac bypass procedures or during cardiac arrestwhen the heart, once prevented from receiving blood, begins to reperfuseand these methods involve administration of the compounds andcompositions of the present invention preferably prior to, orimmediately subsequent to reperfusion, such that reperfusion injury isprevented, treated or reduced. The present invention also providesmethods of preventing and/or treating vascular stroke, cardiovasculardisorders

In another embodiment, the present invention provides in vitro or invivo methods to extend or increase the lifespan and/or proliferationcapacity of cells and thus also methods to treat and/or prevent diseasesassociated therewith and induced or exacerbated by cellular senescenceincluding skin aging, atherosclerosis, osteoarthritis, osteoporosis,muscular dystrophy, degenerative diseases of skeletal muscle involvingreplicative senescence, age-related muscular degeneration, immunesenescence, AIDS and other immune senescence diseases, and otherdiseases associated with cellular senescence and aging, as well as toalter the gene expression of senescent cells.

In a further embodiment, the present invention provides methods oftreating or preventing or ameliorating the effect of cancer and/or toradiosensitize hypoxic tumor cells to render the tumor cells moresusceptible to radiation therapy and thereby to prevent the tumor cellsfrom recovering from potentially lethal damage of DNA after radiationtherapy. A method of this embodiment is directed to specifically andpreferentially radiosensitizing tumor cells rendering the tumor cellsmore susceptible to radiation therapy than non-tumor cells.

In yet another embodiment the present invention provides methods ofpreventing and/or treating vascular stroke, cardiovascular disorders; totreat other conditions and/or disorders such as age-related musculardegeneration, AIDS and other immune senescence diseases, arthritis,atherosclerosis, cachexia, cancer, degenerative diseases of skeletalmuscle involving replicative senescence, diabetes, head trauma, spinalchord injury, immune senescence, inflammatory bowel disorders (such ascolitis and Crohn's disease), acute pancreatitis, mucositis, hemorrhagicshock, splanchnic artery occlusion shock, multiple organ failure (suchas involving any of the kidney, liver, renal, pulmonary, retianl,pancreatic and/or skeletal muscles systems), acute autoimmunethyroiditis, muscular dystrophy, osteoarthritis, osteoporosis, chronicand/or acute pain (such as neuropathic pain), renal failure, retinalischemia, septic shock (such as endotoxic shock), local and/or remoteentothelial cell dysfunction (such are recognized by endo-dependentrelaxant responses and up-regulation of adhesion molecules),inflammation and skin aging.

In one embodiment of the present invention, a person diagnosed withacute retinal ischemia or acute vascular stroke is immediatelyadministered parenterally, either by intermittent or continuousintravenous administration, a compound of any of formulas I, II, III, IVor V either as a single dose or a series of divided doses of thecompound. After this initial treatment, and depending on the person'spresenting neurological symptoms, the person optionally may receive thesame or a different compound of the invention in the form of anotherparenteral dose. The compound of the invention can be admnistered byintermittent or continuous administration via implantation of abiocompatible, biodegradable polymeric matrix delivery system containinga compound of formula I, II, III, IV or V, or via a subdural pumpinserted to administer the compound directly to the infarct area of thebrain.

In a further embodiment, the present invention provides methods toextend the lifespan and proliferative capacity of cells, such as, forexample, in using the compounds of the invention as general mediators inthe generation of oxidants, proinflammatory mediators and/or cytokines,and/or general mediators of leukocyte infiltration, calcium ionoverload, phospholipid peroxidaion, impaired nitric oxide metabolismand/or reduced ATP production

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Shows the distribution of the cross-sectional infarct area atrepresentative levels along the rostrocaudal axis, as measured from theinteraural line in non-treated animals and in animals treated with 10mg/kg of 3,4-dihydro-5-1-(1-piperidinyl)-botoxyl]-1 (2H)-isoquinolinone.

FIG. 2. Shows the effect of intraperitoneal adninistration of3,4-dihydro-5-1-(1-piperidinyl)-butoxy]-1(2H)-isoquinolinone on theinfarct volume.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to compounds, pharmaceutical compositionscontaining the same. methods of using the same, and process of makingthe same, wherein such compounds are useful as inhibitors ofpoly(ADP-ribose) polymerase (PARP). As such, they treat or preventneural tissue damage resulting from cell damage or death due to necrosisor apoptosis, cerebral ischemia and reperfusion injury orneurodegenerative diseases in an animal; they extend the lifespan andproliferative capacity of cells and thus can be used to treat or preventdiseases associated therewith; thev alter gene expression of senescentcells; and they radiosensitize hypoxic tumor cells. Preferably, thecompounds of the invention treat or prevent tissue damage resulting fromcell damage or death due to necrosis or apoptosis, and/or effectneuronal activity, either mediated or not mediated by NMDA toxicity.These compounds are thought to interfere with more than the glutamateneurotoxicitv and NO-mediated biological pathways. Further, thecompounds of the invention can treat or prevent other tissue damagerelated to PARP activation.

For example, the compounds of the invention can treat or preventcardiovascular tissue damage resulting from cardiac ischemia orreperfusion injury. Reperfusion injury, for instance, occurs at thetermination of cardiac bypass procedures or during cardiac arrest whenthe heart, once prevented from receiving blood, begins to reperfuse.

The compounds of the present invention can also be used to extend orincrease the lifespan or proliferation of cells and thus to treat orprevent diseases associated therewith and induced or exacerbated bycellular senescence including skin aging, atherosclerosis,osteoarthritis, osteoporosis, muscular dystrophy, degenerative diseasesof skeletal muscle involving replicative senescence, age-relatedmuscular degeneration, immune senescence, AIDS and other immunesenescence diseases, and other diseases associated with cellularsenescence and aging, as well as to alter the gene expression ofsenescent cells. These compounds can also be used to treat cancer and toradiosensitize hypoxic tumor cells to render the tumor cells moresusceptible to radiation therapy and to prevent the tumor cells fromrecovering from potentially lethal damage of DNA after radiationtherapy, presumably by their ability to prevent DNA repair. Thecompounds of the present invention can be used to prevent or treatvascular stroke; to treat or prevent cardiovascular disorders; to treatother conditions and/or disorders such as age-related musculardegeneration, AIDS and other immune senescence diseases, arthritis,atherosclerosis, cachexia, cancer, degenerative diseases of skeletalmuscle involving replicative senescence, diabetes, head trauma, immunesenescence, inflammatory bowel disorders (such as colitis and Crohn'sdisease), muscular dystrophy, osteoarthritis, osteoporosis, chronicand/or acute pain (such as neuropathic pain), renal failure, retinalischemia, septic shock (such as endotoxic shock), and skin aging.

Preferably, the compounds of the invention act as PARP inhibitors totreat or prevent tissue damage resulting from cell death or damage dueto necrosis or apoptosis; to treat or prevent neural tissue damageresulting from cerebral ischemia and reperfusion injury orneurodegenerative diseases in an animal; to extend and increase thelifespan and proliferative capacity of cells: to alter gene expressionof senescent cells; and to radiosensitize tumor cells.

Another especially preferred embodiment of the invention is apharmaceutical composition which comprises (i) a therapeuticallyeffective amount of the compound of formula I, II or III; and (ii) apharmaceutically acceptable carrier.

As used herein, “alkyl” means a branched or unbranched saturatedhydrocarbon chain comprising a designated number of carbon atoms. Forexample, C₁-R₆ straight or branched alkyl hydrocarbon chain contains 1to 6 carbon atoms, and includes but is not limited to substituents suchas methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl,n-pentyl, n-hexyl, and the like, unless otherwise indicated.

“Alkenyl” means a branched or unbranched unsaturated hydrocarbon chaincomprising a designated number of carbon atoms. For example, C₂-C₆straight or branched alkenyl hydrocarbon chain contains 2 to 6 carbonatoms having at least one double bond, and includes but is not limitedto substituents such as ethenyl, propenyl, isopropenyl, butenyl,iso-butenyl, tert-butenyl, n-pentenyl, n-hexenyl, and the like, unlessotherwise indicated.

“Alkoxy”, means the group —OR wherein R is alkyl as herein defined.Preferably, R is a branched or unbranched saturated hydrocarbon chaincontaining 1 to 6 carbon atoms.

“Cyclo”, used herein as a prefix, refers to a structure characterized bya closed ring.

“Halo” means at least one fluoro, chloro, bromo, or iodo moiety, unlessotherwise indicated.

“Amino” compounds include amine (NH₂) as well as substituted aminogroups comprising alkyls of one through six carbons.

“Ar”, “aryl” or “heteroaryl” means a moiet, which is substituted orunsubstituted, especially a cyclic or fused cyclic ring and includes amono-, bi-, or tricyclic, carbo- or heterocyclic ring, wherein the ringis either unsubstituted or substituted in one to five position(s) withhalo, haloalkyl, hydroxyl, nitro, trifluoromethyl, C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, Cl-C₆alkoxy, C₂-C₆ alkenyloxy, phenoxy, benzyloxy, amino, thiocarbonyl,ester, thioester, cyano, imino, alkylainino, aminoalkyl, sulfhydryl,thioalkyl, and sulfonyl, wherein the individual ring sizes arepreferably 5-8 members; wherein the heterocyclic ring contains 1-4heteroatom(s) selected from the group consisting of O, N, or S, whereinaromatic or tertiary alkyl amines are optionally oxidized to acorresponding N-oxide. Heteroaryls may be attached to other rings orsubstituted through the heteroatom and/or carbon atom of the ring.Particularly preferred aryl or heteroaryl moieties include but are notlimited to phenyl, benzyl, naphthyl, pyrrolyl, pyrrolidinyl, pyridinyl,pyrimidinyl, purinyl, quinolinyl, isoquinolinyl, furyl, thiophenyl,imidazolyl, oxazolyl, thiazolyl, pyrazolyl, and thienyl,

“Phenyl” includes all possible isomeric phenyl radicals, optionallymonosubstituted or multi-substituted with substituents selected from thegroup consisting of amino, trifluoromethyl, C₁-C₆ straight or branchedchain alkyl, C₂-C₆ straight or branched chain alkenyl, carbonyl,thiocarbonyl, ester, thioester, alkoxy, alkenoxy, cyano, nitro, imino,alkylamino, aminoalkyl, sulflhydryl, thioalkyl, sulfonyl, hydroxy, halo,haloalkyl, NR₂ wherein R₂ is selected from the group consisting ofhydrogen, (C₁-C₆)-straight or branched chain alkyl, (C₃-C₆) straight orbranched chain alkenyl or alkynyl, and (C₁-C₄) bridging alkyl whereinsaid bridging alkyl forms a heterocyclic ring starting with the nitrogenof NR₁ and ending with one of the carbon atoms of said alkyl or alkenylchain, and wherein said heterocyclic ring is optionally fused to an Argroup.

Cycloalkyl optionally containing at least one heteroatom includessaturated C₃-C₈ rings, preferably C₅ or C₆ rings, wherein at 1-4heteroatoms selected from O, N or S may be optionally substituted for acarbon atom of the ring. Cycloalkyls optionally containing at least oneheteroatom, as described above, may be substituted by or fused to atleast one 5 or 6 membered aryl or heteroaryl. Prefered cycloalkylscontaining a heteroatom include pyrrolidinyl, imidazolidinyl,pyrazolidinyl, piperidinyl, piperazinyl, morpholino and thiomorpholino.

The compounds of the present invention possess one or more asymmetriccenter(s) and thus can be produced as mixtures (racemic and non-racemic)of stereoisomers, or as individual enantiomers or diastereomers. Theindividual stereoisomers may be obtained by using an optically activestarting material, by resolving a racemic or non-racemic mixture of anintermediate at some appropriate stage of the synthesis, or byresolution of the compound of any of formulas I, II, III, IV and V. Itis understood that the individual stereoisomers as well as mixtures(racemic and non—racemic) of stereoisomers are encompassed by the scopeof the present invention. The S-stereoisomer at atom 1 of formula (I) ismost preferred due to its greater activity.

“Isomers” are different compounds that have the same molecular formulaand includes cyclic isomers such as (iso)indole and other isomeric formsof cyclic moieties. “Stereoisomers” are isomers that differ only in theway the atoms are arranged in space. “Enantiomers” are a pair ofstereoisomers that are non-superimposable mirror images of each other.“Diastereoisomers” are stereoisomers which are not mirror images of eachother. “Racemic mixture” means a mixture containing equal parts ofindividual enantiomers. “Non-racemic mixture” is a mixture containingunequal parts of individual enantiomers or stereoisomers.

The compounds of the invention are useful in a free base form, in theform of pharmaceutically acceptable salts, pharmaceutically acceptablehydrates, pharmaceutically acceptable esters, pharmaceuticallyacceptable solvates, pharmaceutically acceptable prodnigs,pharmaceutically acceptable metabolites, and in the form ofpharmaceutically acceptable stereoisomers. These forms are all withinthe scope of the invention. In practice, the use of these forms amountsto use of the neutral compound.

“Pharmaceutically acceptable salt”, “hydrate”, “ester” or “solvate”refers to a salt, hydrate, ester, or solvate of the inventive compoundswhich possesses the desired pharmacological activity and which isneither biologically nor otherwise undesirable. Organic acids can beused to produce salts, hydrates, esters, or solvates such as acetate,adipate, alginate, aspartate, benzoate, benzenesulfonate,p-toluenesulfonate, bisulfate, sulfamate, sulfate, naphthylate,butyrate, citrate, camphorate, camphorsulfonate,cyclopentane-propionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptanoate, glycerophosphate, hemisulfate heptanoate,hexanoate, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, tosylate and undecanoate.Inorganic acids can be used to produce salts, hydrates, esters, orsolvates such as hydrochloride, hydrobromide, hydroiodide, andthiocyanate.

Examples of suitable base salts, hydrates, esters, or solvates includehydroxides, carbonates, and bicarbonates of ammonia, alkali metal saltssuch as sodium, lithium and potassium salts, alkaline earth metal saltssuch as calcium and magnesium salts, aluminum salts, and zinc salts.

Salts, hydrates, esters, or solvates may also be formed with organicbases. Organic bases suitable for the formation of pharmaceuticallyacceptable base addition salts, hydrates, esters, or solvates of thecompounds of the present invention include those that are non-toxic andstrong enough to form such salts, hydrates, esters, or solvates. Forpurposes of illustration, the class of such organic bases may includemono-, di-, and trialkylamines, such as methylamine, dimethylamine,triethylamine and dicyclohexylamine; mono-, di- ortrihydroxyalkylamines, such as mono-, di-, and triethanolamine, aminoacids, such as arginine and lysine; guanidine; N-methyl-glucosamine:N-methyl-glucamine; L-glutamine; N-methyl-piperazine; morpholine:ethylenediamine; N-benzyl-phenethylamine:(trih,ydroxy-methyl)aminoethane: and the like. See, for example,“Pharmaceutical Salts,” J. Pharm. Sci., 66:1, 1-19 (1977). Accordingly,basic nitrogen-containing groups can be quaternized with agentsincluding: lower alkyl halides such as methyl, ethyl, propyl, and butylchlorides, bromides and iodides; dialkyl sulfates such as dimethyl,diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides and iodides; andaralkyl halides such as benzyl and phenethyl bromides.

The acid addition salts, hydrates, esters, or solvates of the basiccompounds may be prepared either by dissolving the free base of a PARPinhibitor of the present invention in an aqueous or an aqueous alcoholsolution or other suitable solvent containing the appropriate acid orbase, and isolating the salt by evaporating the solution. Alternatively,the free base of the PARP inhibitor of the present invention can bereacted with an acid, as well as reacting the PARP inhibitor having anacid group thereon with a base, such that the reactions are in anorganic solvent, in which case the salt separates directly or can beobtained by concentrating the solution.

“Pharmaceutically acceptable prodrug” refers to a derivative of theinventive compounds which undergoes biotransformation prior toexhibiting its pharmacological effect(s). The prodrug is formulated withthe objective(s) of improved chemical stability, improved patientacceptance and compliance, improved bioavailability, prolonged durationof action, improved organ selectivity, improved formulation (e.g.,increased hydrosolubility), and/or decreased side effects (e.g.,toxicity). The prodrug can be readily prepared from the inventivecompounds using methods known in the art, such as those described byBurger's Medicinal Chemistry and Drug Chemistry, Fifth Ed., Vol. 1, pp.172-178, 949-982 (1995). For example, the inventive compounds can betransformed into prodrugs by converting one or more of the hydroxy orcarboxy groups into esters.

“Pharmaceutically acceptable metabolite” refers to drugs that haveundergone a metabolic transformation. After entry into the body, mostdrugs are substrates for chemical reactions that may change theirphysical properties and biologic effects. These metabolic conversions,which usually affect the polarity of the compound, alter the way inwhich drugs are distributed in and excreted from the body. However, insome cases, metabolism of a drug is required for therapeutic effect. Forexample, anticancer drugs of the antimetabolite class must be convertedto their active forms after they have been transported into a cancercell. Since most drugs undergo metabolic transformation of some kind,the biochemical reactions that play a role in drug metabolism may benumerous and diverse. The main site of drug metabolism is the liver,although other tissues may also participate.

The term “neurodegenerative diseases” includes Alzheimer's disease,Parkinson's disease and Huntington's disease.

The term “nervous insult” refers to any damage to nervous tissue and anydisability or death resulting therefrom. The cause of nervous insult maybe metabolic, toxic, neurotoxic, iatrogenic, thermal or chemical, andincludes without limitation, ischemia, hypoxia, cerebrovascularaccident, trauma, surgery, pressure, mass effect, hemmorrhage,radiation, vasospasm, neurodegenerative disease, infection, Parkinson'sdisease, amyotrophic lateral sclerosis (ALS), myelination/demyelinationprocess, epilepsy, cognitive disorder, glutamate abnormality andsecondary effects thereof.

The term “neuroprotective” refers to the effect of reducing, arrestingor ameliorating nervous insult, and protecting, resuscitating, orreviving nervous tissue that has suffered nervous insult.

The term “preventing neurodegeneration” includes the ability to preventneurodegeneration in patients diagnosed as having a neurodegenerativedisease or who are at risk of developing a neurodegenerative disease.The term also encompasses preventing further neurodegeneration inpatients who are already suffering from or have symptoms of aneurodegenerative disease.

The term “treating” refers to:

(i) preventing a disease, disorder or condition from occurring in ananimal that may be predisposed to the disease, disorder and/orcondition, but has not yet been diagnosed as having it:

(ii) inhibiting the disease, disorder or condition, i.e., arresting itsdevelopment; and

(iii) relieving the disease, disorder or condition, i.e., causingregression of the disease, disorder and/or condition.

The term “neural tissue damage resulting from ischemia and reperfusioninjury and neurodegenerative diseases” includes neurotoxicity, such asseen in vascular stroke and global and focal ischemia.

A feature charactenstic of many of these transformations is that themetabolic products are more polar than the parent drugs, although apolar drug does sometimes yield a less polar product. Substances withhigh lipid/water partition coefficients, which pass easily acrossmembranes, also diffuse back readily from tubular urine through therenal tubular cells into the plasma. Thus, such substances tend to havea low renal clearance and a long persistence in the body. If a drug ismetabolized to a more polar compound, one with a lower partitioncoefficient, its tubular reabsorption will be greatly reduced. Moreover,the specific secretory mechanisms for anions and cations in the proximalrenal tubules and in the parenchymal liver cells operate upon highlypolar substances.

As a specific example; phenacetin (acetophenetidin) and acetanilide areboth mild analgesic and antipyretic agents, but are each transformedwithin the body to a more polar and more effective metabolite,p-hydroxyacetanilid (acetaminophen), which is widely used today. When adose of acetanilid is given to a person, the successive metabolites peakand decay in the plasma sequentially. During the first hour, acetanilidis the principal plasma component. In the second hour, as the acetanilidlevel falls, the metabolite acetaminophen concentration reaches a peak.Finally, after a few hours, the principal plasma component is a furthermetabolite that is inert and can be excreted from the body. Thus, theplasma concentrations of one or more metabolites, as well as the drugitself, can be pharmacologically important.

The reactions involved in drug metabolism are often classified into twogroups, as shown in the Table II. Phase I (or functionalization)reactions generally consist of (1) oxidative and reductive reactionsthat alter and create new functional groups and (2) hydrolytic reactionsthat cleave esters and amides to release masked functional groups. Thesechanges are usually in the direction of increased polarity.

Phase II reactions are conjugation reactions in which the drug, or oftena metabolite of the drug, is couple to an endogenous substrate, such asglucuronic acid, acetic acid, or sulfuric acid.

TABLE II Phase I Reactions (functionalization reactions): (1) Oxidationvia the hepatic microsomal P450 system: Aliphatic oxidation Aromatichydroxylation N-Dealkylation O-Dealkylation S-Dealkylation EpoxidationOxidative deamination Sulfoxide formation Desulfuration N-Oxidation andN-hydroxylation Dehalogenation (2) Oxidation via nonmicrosomalmechanisms: Alcohol and aldehyde oxidation Purine oxidation Oxidativedeamination (monoamine oxidase and diamine oxidase) (3) Reduction: Azoand nitro reduction (4) Hydrolysis: Ester and amide hydrolysis Peptidebond hydrolysis Epoxide hydration Phase II Reactions (conjugationreactions): (1) Glucuronidation (2) Acetylation (3) Mercapturic acidformation (4) Sulfate conjugation (5) N—, O—, and S—methylation (6)Trans-sulfuration

The compounds of the present invention exhibit pharmacological activityand are, therefore, useful as pharmaceuticals. In particular, thecompounds exhibit central nervous and cardiac vesicular system activity.

It is understood that tautomeric forms, when possible, are included inthe invention. For example, the tautomeric forms of the followingcompounds are exemplary:

Many of the PARP inhibitors are known and, thus, can be synthesized byknown methods from starting materials that are known, may be availablecommercially, or may be prepared by methods used to preparecorresponding compounds in the literature. See, for example, Suto etal., “Dihydroiso-quinotinones: The Design and Synthesis of a New Seriesof Potent Inhibitors of Poly(ADP-ribose) Polymerase”, Anticancer DrugDes., 6:107-17 (1991), which discloses processes for synthesizing anumber of different PARP inhibitors.

Typically, the PARP inhibitors used in the composition of the inventionwill have an IC₅₀ for inhibiting poly(ADP-ribose) polymerase in vitro ofabout 20 μM or less, preferably less than about 10 μM, more preferablyless than about 1 μM, most preferably less than about 0.1 μM.

The PARP inhibitor3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone, forexample, has been reported to inhibit PARP with an IC₅₀ of 40 nM by Sutoet al., cited above.

There are multiple routes which may be undertaken to prepare thecompounds of the present invention. Two of these routes for thepreparation of the xanthene derivatives of this invention aredemonstrated below by schemes 1-3 and 4-7.

The xanthene ring may be generically substituted as set forth in formulaI. Such xanthene starting derivatives are known in the chemistryliterary and are accessible by processes known to one skilled in theart. The process sequence set forth herein does not present an exactsequence of reactions by which the compound must be made; that is, thesequence of reactions can be rearranged in several ways to reach thetarget molecule.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The xanthene and thioxanthene derivatives of this invention arerepresented by previously defined formulas I-V. As an example, thexanthene derivatives of this invention can be prepared in a conventionalmanner as illustrated below by Schemes 1-5. The xanthene ring may begenerically substituted as set forth in formula I. Such xanthenestarting derivatives are known in the chemistry literature andaccessible by processes known to one skilled in the art. The processsequence set forth herein does not present an exact sequence ofreactions by which the compound must be made; that is, the sequence ofreactions can be rearranged in several ways to reach the targetmolecule. Scheme 1 below illustrates schematically the preparation ofcompounds Example 1 through Examples 4.

Step 1

9-Arninomehtylxanthene 2 is available by reduction of 9-carboxamide 1using sodium boronhydride in dioxane. Other reduction methods can besubstituted include lithium aluminum hydride and other boronhydrides.The solvent can also be varied, DMSO, tetrahydrofuran, diethylether andother organic solvent can be used. The temperature of the reaction isgeneral between 0° C. and 200° C. For example, 9-aminomethylxanthene canbe made as follows: To a stiffed suspension of sodium boronhydride (1.89g, 50 mmol) and 9-xanthenecarboxamide (2.25g, 10 mmnol) in dioxane (20mL) is added acetic acid (3.0 g, 50 mmol) in dioxane (10 mL) over aperiod of 10 minutes at 10° C. The reaction mixture is stiffed at refluxfor 2 hours and concentrated to dryness in vacuo. The excess reductivereagent is decomposed with water and the mixture is extracted withchloroform. The chloroform layer is washed with water, dried overanhydrous sodium sulfate and evaporated in vacuo. The residue ispurified by silica gel column chromatography (ethylacetate:methanol, 9:1as eluant) to give a white solid (1.6 g, 7.6 mmol) in 76.2% yield.

Step 2

The 9-isocyanomehtylxanthenes 3 is obtained by condensation of the aminogroup with phosgene in a heated solution of toluene. Other solvents.such as p-nitrobenzene, can also be used. The new formed isocyanofunctionality serves as an electrophile for intermolecularFriedle-Crafts reaction in next step. Other functionalities includeN-carbonylimidazole, N-carbonylbenzotriazole and N-ethytformnate canalso be applied in this type of reactions. These functionalities can beformed by reactions of the 9-aminomethylxanthenes 2 withcarbonyldmiidazole, carbonyldibenzotriarole and ethyl chioroformaterespectively.

Step 3

The desired xanthane product,3,4-dihydrobenzopyrano[4,3,2-de]isoquinoli-1-one 4 can be obtained by anintramolecular Friedle-Crafts acylation using acid as catalyst. Zincchloride, aluminum chloride, titanium (IV) chloride, hydrochloric acidor acetic acid may be used, but polyphosphoric acid is often preferredfor this type of intramolecular cycloadditions. The temperature can varyfrom 0-150° C. For example,3,4-dihydrobenzopyrano[4,3,2-de]isoquinolin-1-one 4 can be prepared asfollows: To a liquid of 12 g of polyphosphoric acid in a 500 mL beakeris added 9-xanthenemethylisocyanide 3 (2.37 g, 10 mmol) portion wisewith manual stirring at 90° C. The mixture is stirred for three minutesand then 100 g of more polyphosphoric acid is added. Vigorous stirringis applied for four minutes when temperature is kept at 90° C. Themixture is allowed to cool to 60° C. and 40 g of crushed ice is addeduntil the polyphophoric acid is completely hydrolyzed and a brown solidis separated. The solid is collected by vacuum filtration and thenrecrystallized in chloroform to afford a unsaturated product (0.15 g,0.63 mmol) in 6.3% yield.

The solid is dissolved in glacial acetic acid (100 mL) and the solutionis placed in a hydrogenation bomb. Palladium (10% on carbon. 500 mg) isadded. The bomb is set pressure at 2000 psi. The mixture of the contentis stirred for 20 hours under the pressure and poured through a flutedfilter paper to remove the catalyst. The solvent of the filtrate isremoved in vacuo to give a yellow solid, which is recrystallized inchloroform to afford desired product (0.95 g, 4.0 mmol) in 80% yield.

Monosulfonation of 3,4-dihydrobenzopyrano[4,3,2-de]isoquinoli-1-one 4can be achieved using chorosulfonic acid neat to give compound 5. Thetemperature can vary from −20 to 100° C. Amidation of compound 5 can becarried out by a variety of conditions known to those skilled in theart, including reaction with first or secondary amines using pyridine ortriethyl amine as base. Typical solvents include chlorinated solvents,various ethers, and dipolar aprotic solvents like DMF. Thus finalproducts of compounds 6 can be prepared from3,4-dihydrobenzopyrano[4,3,2-de]isoquinoli-1-one 4 using Generalprocedure A and B.

Scheme 2 below illustrates schematically the preparation of compoundsExample 5 through Example 9.

Step 4

Preparation of 3-phenoxyphthalonitrile 7. The starting materials3-nitrophthalonitrile 7 and or p-substituted phenoxide 8 are eitherreadily available from commercial sources or can be prepared by knownmethods by those skilled in the art. The desired compound can be easilyobtained by coupling the orthodinitrile with the phenoxide salt indipolar aprotic solvent, such as DMF, low dialkyl ketones, lower alkylnitriles. Temperature range can be 0-150° C. For example, a mixture of0.1 Mol of 3-nitrophthalonitrile and 0.14 Mol of sodium phenoxide (orp-substituted phenoxide) in 120 ml of dry DMF is mechanically stirredfor 2.5 hours at room temperature and then poured into water. Theprecipitate is collected by filtration, washed with water, and driedunder vacuum to give 19 g (0.086 Mol. 86%) of the compound 9, mp110-112° C.

Step 5

Preparation of 9-oxoxanthene-1-carboxylic acid 10. The formation oforthocarboxylic acid groups from the cyano groups can be achieved byhydrolysis of the aryl nitrile with either mineral acids, includesulfuric acids and hydrochloric acids, or strong inorganic base. Theseconditions are understood by those skilled in the art. An intramolecularFriedle-Crafts acylation can occur as soon as the diacids are formed toafford 9-oxoxanthene-1-carboxylic acid 10. For example, a solution of0.1 Mol of 3-phenoxyphthalonitrile is dissolved in 150 ml of a mixtureof 60 per cent sulfuric acid and glacial acid. The mixture is refluxedfor 6 hours and then poured into 1000 mL of ice-cold water. pH value ofthe suspension is adjusted to 10 with sodium carbonate. Impurities areextracted out by partitioning of the basified solution with ethylacetate. Precipitation is formed upon adding hydrogen chloride (to pH5)to the aqueous solution, and collected by filtration to give a designedacid in 70% yield.

Step 6

Preparation of 9-oxoxanthene-1-carboxylic acid methyl ester 11.

Esterification of acid can be achieved by several conventional methodsby those skilled in the art. These procedures include utilizingdiazomethane or methyl alcohol catalyzed by mineral acids. For example,ethereal diazomethane solution can be prepared fromN-methyl-N-nitrosotoluene-p-sulphonamide and potassium hydroxide from awater bath. 9-Oxoxanthene-1-carboxylic acid (1.29 g, 5 mmol) in absolutemethanol is added to this ethereal diazomethane solution at 0° C. untila pale yellow color formed. Removal of the solvent provides the desiredester solid in 100% yield. Thus the final products, Examples 5-9, can beprepared by condensation hydrazine with the ketonester 11 as describedin General procedure C.

Scheme 3 below illustrates schematically the preparation of compoundsExample 10 through Example 11.

Step 7

Preparation of 7-amino-9-oxoxanthene-1-carboxylic acid methyl ester 12.Reduction of the nitro group can be carried out under catalytichydrogenation conditions utilizing metal catalysts described inaldrichimica Acta 1979:12, or with hydrazine, metal hydride reducingagents such as NaBH₄, B₂H₆, and the like. Suitable solvents includewater, lower alcohol, ethers, THF, lower alkyl organic acids such asacetic acid. Temperature can vary from 0-100° C. Desired final products,Examples 11-12, can be prepared by condensing the ketonester 12 withhydrazine using General procedure C, followed by an amide bond formationof the phalazine derivative 13 using General procedure F.

Scheme 4 below illustrates schematically the preparation of Example 12through Examples 24.

Step 9

Preparation of 7-bromomethyl-9-oxoxanthene-1-carboxylic acid methylester 14. Brominating agents include N-bromosuccinimide, bromine,complexed bromine such as pyridinium bromide, perbromide, and the likecan be used to convert 7-methyl-9-oxoxanthene-1-carboxylic acid methylester 11 to 7-bromomethyl-9-oxoxanthene-1-carboxylic acid methyl ester14. Solvents include chlorinated hydrocarbones, dipolar aproticsolvents, and various ethers. Temperature can range from 0-100° C. Forexample, a suspension of 7-methyl-9-oxoxanthene-1-carboxylic acid methylester (0.1 Mol), N-bromosuccinimide (0.12 Mol) and benzoylperoxide (10mg) in dry carbon tetrachloride (300 mL) is stirred at 60° C. for 6hours. The mixture is filtered, the solid is washed successively withsmall amounts of chloroform, water and ether, and then dried to leave adesired product as white solid.

Displacement of the bromo group of compound 14 with nucleophiles such asamine or alcohol, thiol using General procedure D or E provides thecompound 15. The ketonester 15 can be cyclized with hydrazine usingGeneral procedure C to give desired final products, Example 12-24.

Scheme 5 below illustrates schematically the preparation of Example 26through Example 29.

Monosulfonation of the ketone ester 11 to give compound 16 can beachieved using neat chorosulfonic acid to react with the substrate 11.The temperature can vary from −20 to 100° C. Amidation of compound 16can be carried out by a variety of conditions known to those skilled inthe art, including reaction with first or secondary amines usingpyridine or triethyl amine as base. Typical solvents include chlorinatedsolvents, various ethers, and dipolar aprotic solvents like DMF. Thusfinal product of compounds can be prepared from compound 17 andhydrazine as described in General procedure C.

General Procedure A

To a liquid of chlorosulfonic acid (10 mL) is added the compound 5 orketone ester (11, 7.87 mmol) under nitrogen at 0° C. The resultingmixture is allowed to warm to room temperature, continuously stirred forabout 2 days and poured onto 100 g of ice. The residue is collected byfiltration and washed with water, ethanol 15 to afford the product 5 or16 as solid in >80% of yield.

General Procedure B

To a suspension of the sulfonyl chloride 5 or (16, 5.67 mmol) inmethylene chloride (100 mL) is added triethyl amine (1.6 mL) and aminecompound (5 mmol) under nitrogen at 0° C. The reaction mixture isallowed to warm to room temperature, stirred continuously for 1 hour andpoured into 100 mL of water. The organic layer is collected, washed withwater, brine and concentrated in vacuo. The product is purified viacrystallization or silica column chromatography using methanol/methylenechloride as eluent to afford solid product in 60-90% of yield.

General Procedure C

A benzopyrano[4,3,2-delphthalazine ring can be formed by condensation ofthe ketone ester with hydrazine. To a solution of the ketonester 11 or15, (17, 5 mmol) in absolute ethanol (10 mL) is added anhydroushydrazine in ethanol (1 mL) drop wise at room temperature. The solutionis refluxed for overnight and cooled to room temperature. Ice-cold water(100 mL) is added and brown solid is separated. The solid is collectedby vacuum filtration and washed with water, small amount of methanol togive a solid product in 10-80% of yield.

General Procedure D

To a solution of the bromo compound (14, 10 mmol) in dry DMF (100 mL) isadded potassium carbonate (100 mmol) and secondary amine (10 mmol). Thereaction mixture is heated to 70° C. for 6 hours and cooled to 0° C.Water (100 mL) is added to the reaction mixture, followed by ethylacetate (200 mL). The organic layer is collected, washed with water,brine and dried over sodium sulfate. The solvent is removed in vacuo.The residue is purified by column chromatography on silica gel usingethyl acetate /hexane as eluent to give the product 15 in 60-90% ofyield.

General Procedure E

To a stirred solution of the bromo compound 14 (10 mmol) and phenolderivative or thiol derivative (10 mmol) in acetone (150 mL) is addedpotassium carbonate (100 mmol). The reaction mixture is heated to refluxfor 6 hours. The solvent is then removed in vacuo, and water (100 mL)and ethylacetate (100 mL) are added to the residue. The organic layer iscollected, washed with water, brine and dried over sodium sulfate. Thesolvent is removed in vacuo. The residue is purified by columnchromatography on silica gel using ethyl acetate/hexane as eluent togive the product 15 in 60-80% of yield.

General Procedure F

To a suspension of compound phthalazine derivative 13 (10 mmol) in dryDMF (20 mL) is added succinic or acetic anhydride (11 mmol). The mixtureis heated to 110° C. for 3 hours and cooled to room temperature. Themixture is continuously stirred for 2 hours after addition of THF (100mL). The solid is collected by filtration, washed with methanol toafford a desired product without further purification.

Other manners, variations or sequences of preparing the compounds ofFormula I will be readily apparent to those skilled in the art.

The compounds of formula I may be useful in the free base form, in theform of base salts where possible, and in the form of addition salts, aswell as in the free acid form. All these forms are within the scope ofthis invention. In practice, use of the salt form amounts to use of thebase form. Pharmaceutically acceptable salts within the scope of thisinvention are those derived from mineral acids such as hydrochloric acidand sulfuric acid; and organic acids such as ethanesulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid, and the like, giving thehydrochloride, sulfonate, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, and the like respectively, or those derived frombases such as suitable organic and inorganic bases. Examples ofpharmaceutically acceptable base addition salts with compounds of thepresent invention include organic bases which are nontoxic and strongenough to form such salts. These organic bases and the use thereof arereadily understood by those skilled in the art. Merely for the purposeof illustration, such organic bases may include mono-, di-, andtrialkylamines, such as methylamine, diethylamine and triethylamine;mono-, di-, or trihydroxyalkylamines such as mono-, di-, andtriethanolamine; amino acids such as arginine, and lysine; guanidine;N-methylglucosamine; N-methylgiucamine. L-glutamine; N-methylpiperazine;morpholine; ethylenedianane; N-benzylphenethylamine;tris(hydroxymethyl)antinoethane; and the like.

The acid addition salts of the basic compounds may he prepared bydissolving the free base of the compound of formula I in aqueous oraqueous alcohol solution or other suitable solvents containing theappropriate acid or base and isolating the salt by evaporating thesolution, or by reacting the free base of the compound of formula I withan acid as well as reacting the compound of formula I having an acidgroup thereon with a base such that the reactions are in an organicsolvent, in which case the salt separates directly or can be obtained byconcentration of the solution.

The compounds of this invention contain one or more asymmetric carbonatoms. Therefore, the invention includes the individual stereoisomersand mixtures thereof as well as the racemic compounds. The individualisomers may be prepared or isolated by methods known in the art.

The compounds of the invention exhibit pharmacological activity and are,therefore, useful as pharmaceuticals. In particular the compoundsexhibit central nervous and cardiac vesicular system activity.

EXAMPLE 1

To a solution of 0.3 g of compound 4 in 7.5 mL of concentrate sulfuricacid (98%) is dropwise-added 2.0 mL of fuming sulfuric acid at 0° C. Themixture is stirred for 3 hours at 0° C. and poured into 100 mL ofice-cold water. The precipitation is collected by filtration, washedwith water to give 0.17 g of a whiite solid, mp >300 (dec.). ¹H-NMR (400MHz, DMSO-d6): 8.20(d, 1H, 4Hz); 7.62(d, 1H, 8Hz); 7.53(s, 1H); 7.51(d,1H, 8Hz); 7.41(t, 1H, 8Hz); 7.30(d, 1H, 8.0Hz); 7.09(d, 1H, 8.0Hz);4.50-4.55(dd, 1H, 8Hz); 4.02-3.96(m, 1H); 3.46(t, 1H, 12Hz).

EXAMPLE 2

Prepared from compound 5 and N-(2-aminoethyl)piperidine according toGeneral Procedure B. Purification of compound by crystallization inwater-acetone gives a white solid, mp 265-269° C. ¹H-NMR (400 MHz,DMSO-d6): 8.35(m, 1H), 7.82 (s, 1H), 7.75 (m, 1H), 7.67 (m, 1H),7.46-7.43 (m, 1H), 7.37 (m, 1H), 4.58-4.54 (m, 1H), 4.10 (m, 1H),3.53-3.47 (m, 1H), 3.39 (m, 2H), 3.06 (bs, 2H), 3,02 (bs, 2H), 2.32 (s,2H), 1.44-1.19 (m, 6H).

EXAMPLE 3

Prepared from compound 5 and N-methylpiperazine according to GeneralProcedure B. Purification of compound by crystallization in acetic acidgives a white solid, mp 300° C. (dec.). ¹H-NMR (400 MHz, CDCl₃):7.78-7.84 (d, J=8 Hz, 1 H), 7.58-7.64 (d, J=8 Hz, 1 H), 7.53 (s, 1 H),7.32-7.40 (t,J=8, 16 Hz), 7.16-7.26 (m, 2 H), 6.30 (br s, 1 H),4.36-4.50 (q, J=6, 14, 20Hz, 1 H), 4.02-4.12 (m, 1 H), 3.60-3.74 (t,J=12, 24 Hz, 1 H), 3.01 (br s, 4 H), 2.46 (br s, 4 H), 2.24. (s, 3 H).

EXAMPLE 4

Prepared from compound 5 and N-acetylpiperazine according to GeneralProcedure B. Purification of compound by crystallization in acetic acidgives a white solid (45% yield), mp 290° C. (dec.). ¹H-NMR (400 MHz,CDCl₃): 7.88-7.93 (d, J=8 Hz, 1 H), 7.67-7.72 (dd, J=4, 12 Hz, 1 H),7.62 (s, 1 H), 7.43-7.49 (t, J=8, 16Hz, 1H), 7.27-7.36 (m, 2 H),4.54-4.62 (dd, J=8, 20Hz, 1 H), 4.15-4.25 (m, 1 H), 3.70-3.85 (m, 3 H),3.59(s, 2 H), 3.04 (s, 4 H), 2.06 (s, 3 H).

EXAMPLE 5

Prepared from the compound 11 (R=MF) and hydrazine according to GeneralProcedure C. Purification of compound by crystallization in ethanolgives a whit flake solid (44% yield), mp 332-334° C. ¹H-NMR (400 MHz,DMSO-d₆): 12.5 (s, br, 1H); 7.90-7.87 (m, 3H); 7.69 (dd, J=7.2, 1.9 Hz,1H; 7.38 (dd, J=8.8, 1.9 Hz, 1H); 7.31 (d, J=8.4 Hz, 1H); 2.39 (s, 3H).

EXAMPLE 6

Prepared from the compound 11 (R=F) and hiydrazine according to GeneralProcedure C. Purification of compound by crystallization in ethanolgives a whit solid (20% yield), mp 355° C. (dec.). ¹H-NMR (400 MHz,DMSO-d₆): 12.7(s, br, 1H); 8.42 (d, J=2.0 Hz, 1H); 7.91 (m, 2H);7.73(dd, J=2.0, 6.8 Hz, 1H); 7.41(m, 2H)

EXAMPLE 7

Prepared from the compound 11 (R=t-Bu) and hydrazine according toGeneral Procedure C. Purification of compound by crystallization inethanol gives a solid (30% yield), mp 380° C. (dec.). ¹H-NMR (400 MHz,DMSO-d₆): 8.07 (d, J=2.5 Hz, 1H); 7.91 (dd, J=7.6, 7.0 Hz, 1H); 7.89 (d,J=7.6 Hz, 1H); 7.72 ( dd, J=7.0, 2.0 Hz, 1H); 7.65 (dd, J=9.0, 2.5 Hz,1H); 7.37 (d, J=9.0 Hz, 1H); 1.38 (s, 9H)

EXAMPLE 8

Prepared from the compound 11 (R=NH₂) and hydrazine according to GeneralProcedure C. Purification of compound by crystallization in ethanolgives a yellowish solid (60% yield), mp 300° C. (dec.). ¹H-NMR (400 MHz,DMSO-d₆): 5.29-5.31 (d, 2H, J=7.8 Hz), 6.81-6.83 (dd, 1H, J=8.8, 2.4Hz), 7.14-7.16 (d, 1H, J=8.8 Hz), 7.26-7.27 (d, 1H, J=2.2 Hz), 7.63-7.65(d, 1H, J=7.3 Hz), 7.83-7.89 (dd, J=7.5, 15.5 Hz), 7.85 (s, 1H,),12.52(s, br, 1H).

EXAMPLE 9

Prepared from the compound 11 (R=NO₂) and 1.5 equivalent of hydrazineaccording to General Procedure C. Purification of compound bycrystallization in acetic acid gives a yellowish solid (20% yield),mp >300° C. (dec.). ¹H-NMR (400 MHz, DMSO-d₆): 7.46-7.49 (d, 1H,J=9.2Hz,), 7.75-7.77 (d, 1H, J=8.1 Hz), 7.97-8.01 (dd, 1H, J=8.1 Hz),8.13-8.15 (d, 1H, J=7.9 Hz), 8.38-8.42 (dd, 1H, J=9.5 Hz), 9.07-9.08 (d,1H, J=2.6 Hz).

EXAMPLE 10

Prepared from the amino compound 13 and acetic anhydride according toGeneral Procedure F. Purification of the compound by crystallization inethanol to gives a yellow solid in 80% yield, mp 350° C. (dec.). ¹H-NMR(400 MHz, DMSO-d₆): 2.08 (s, 1H), 7.36-7.38 (d, 1H, J=8.9), 7.66-7.70(dd, 1H, J=2.6, 16.1 Hz), 7.68-7.69 (d, 1H, J=2.2 Hz), 7.88-7.91 (d, 1H,J=8.6 Hz), 7.89 (s, 1H), 8.49 (d, 1H, J=2.5 Hz), 10.15 (s, 1H, NHCO),12.62 (s, 1H).

EXAMPLE 11

Prepared from the amino compound 13 and succinic anhydride according toGeneral Procedure F. Purification of the compound by crystallization inethanol to gives a yellow solid in 20% yield, mp 320° C. (dec.). ¹H-NMR(400 MHz, DMSO-d₆): 2.50-2.60 (m, 4H), 7.45-7.47 (d, 1H, J=8.9 Hz),7.74-7.77 (dd, 1H, J=9.2, 2.5 Hz), 7.76-7.79 (dd, 1H, J=7.2, 2.1),7.96-7.98 (d, 1H, J=9.3 Hz), 7.97 (s, 1H), 8.56-8.57 (d, 1H J=2.5 Hz)10.29(s, 1H), 12.72 (s, 1H).

EXAMPLE 12

Prepared from the compound 14 and cysteamine according to GeneralProcedure E. Purification of compound by crystallization in ethanolgives white solid product in 30% of yield, mp 255-275° C.(methylsulfonic acid salt). ¹H-NMR (400 MNz, DMSO-d₆): 2.29 (s, 3H),2.64 (t, 2H, J=7.3 Hz), 301 (br s, 2H), 3.92 (s, 2H), 7.42 (d, 1H, J=8.5Hz), 7.54-7.57 (dd, 1H, J=2.1 Hz, 8.6 Hz), 7.71-7.76 (m, 4H), 7.89-7.95(m, 2H), 8.0 (d, 1H, J=2.1 Hz), 12.69 (s, 1H).

EXAMPLE 13

Prepared from the compound 14 and 4-mercaptopyridine according toGeneral Procedure E. Purification of compound by crystallization inethanol gives white solid product in 38% of yield, mp 237-240° C. ¹H-NMR(400 MHz, DMSO-d₆): 12.68 (s, 1H), 8.61 (m, 2H), 8.24 (d, 1H, J=2.2 Hz),7.92-7.85 (m, 4H), 7.70 (m, 2H), 7.44 (d, 1H, J=8.5 Hz, 4.70 (s, 2H),2.30 (s, 3H),

EXAMPLE 14

Prepared from the compound 14 and hydrazine according to GeneralProcedure D. Purification of compound by crystallization in acetic acidgives a yellow solid product in 78% of yield, Mp >300° C. ¹H-NMR (400MHz, DMSO-d₆): 12.64 (s, br, 1H), 8.04 (d, J=2 Hz, 1H), 7.93-7.88 (m,2H) 7.71 (dd, J=1.9 Hz and 7.3 Hz, 1H), 7.51 (dd, J=2.1 Hz and 8.5 Hz,1H), 7.37 (d, J=8.4 Hz, 1H), 3.82 (s, 2H).

EXAMPLE 15

Prepared from the compound 14 and potassium cyanide according to GeneralProcedure D. Purification of compound by crystallization in acetic acidgives a light yellow solid product in 70% of yield, mp >300° C. ¹H-NMR(400 MHz, DMSO-d₆): 12.70 (s, 1H, NH), 8.10 (d, J=2.0 Hz, 1H), 7.91 (m,2H), 7.72 (dd, J=1.8, 7.2 Hz, 1H), 7.53 (dd, J=2.2, 8.6 Hz, 1H), 7.46(d, J=8.6Hz, 1H), 4.18 (s, 2H).

EXAMPLE 16

Prepared from the compound 14 and 1-phenylpiperazine according toGeneral Procedure D). Purification of compound by crystallization inethanol gives a white solid product in 89% of yield, mp >300° C. (dec.).¹H-NMR (400 NMz, DMSO)-d₆): 12.63 (s, br, 1H), 8.06 (d, J=1.9 Hz, 1H),7.90 (m, 2H), 7.72 (dd, J=7.1 and 1.9 Hz, 1H), 7.53 (dd, J=8.5 and 2.0Hz, 1H), 7.40 (d, J=8.5, 1H), 7.19 (m, 2H), 6.92 (d, J=8.0 Hz, 2H), 6.76(t, J=7.3 Hz, 1H1), 3.61 (s, 2H), 3,14 (t, J=4.3 Hz, 2H), 2.56 (t, J=4.2Hz, 2H).

EXAMPLE 17

Prepared from the compound 14 and 1-(2-pyridyl)piperazine according toGeneral Procedure D. Purification of compound by crystallization inethanol gives a white solid product in 89% of yield, mp >300° C. (dec.).¹H-NMR (400 MHz, DMSO-d₆): 12.64 (s, br, 1H), 8.09 (m, 2H), 7.90 (m,2H), 7.71 (dd, J=7.2 and 1.8 Hz, 1H), 7.52 (m, 2H), 7.40 (d, J=8.5 Hz,1H), 6.80 (d, J=8.6 Hz, 1H), 6.62 (m, 1H), 3.60 (s, 2H), 3.49 (s, 4H),3.30 (s, 4H).

EXAMPLE 18

Prepared from the compound 14 and piperidine according to GeneralProcedure D. Purification of compound by crystallization in ethanolgives a white solid product in 69% of yield, mp 195-200° C. ¹H-NMR (400MHz, DMSO-d₆): 1.30-1.45 (m, 1 H), 1.53-1.75 (m, 3H), 1.75-1.90 (m, 2H),2.35 (s, 3H), 2.82-3.0 (2H), 3.30-3.4 (m, 2H), 4.39 (d, 2H, J=5.2 Hz),7.51 (d, 1H, J=8.5 Hz), 7.65-7.68 (dd, 1H, J=2.1 Hz, 8.5 Hz), 7.74-7.76(m, 1H), 7.91-7.96 (m, 2H), 8.29 (d, 1H, J=2.1 Hz), 9.33 (bs, 1H), 12.79(s, 1H).

EXAMPLE 19

Prepared from the compound 14 and N-methylbutylamine according toGeneral Procedure D. Purification of compound by crystallization inethanol gives a white solid product in 69% of yield, mp 133-135° C.¹H-NMR (400 MHz, DMSO-d₆): 0.91 (t, 3H, J=7.4 Hz), 1.29-1.34 (m, 2H),1.65-1.70 (m, 2H), 2.35 (s, 3H), 2.69 (d, 3H, J=4.8 Hz), 3.03-3.05 (m,1H), 3.14 (m, 1H), 7.53 (d, 1H, J=8.5 Hz), 7.67-7.69 (dd, 1H, J=2.0 Hz,8.5 Hz), 7.73-7.76 (m, 1H), 7.91-7.96 (m, 2H), 8.30 (s, 1H, J=2.0 Hz),9.50 (s, 1H), 12.79 (s, 1H).

EXAMPLE 20

Prepared from the compound 14 and N-dimetlylamine according to GeneralProcedure D. Purification of compound by crystallization in ethanolgives a white solid product in 70% of yield. ¹H-NMR (400 MHz, DMSO-d₆):2.36 (s, 3H), 2.78 (d, 6H, J=4.8 Hz), 4.40 (m, 2H), 7.53 (d, 1H, J =8.5Hz), 7.65-7.70 (m, 1H), 7.75 (m, 1H), 7.92-7.95 (m, 2H), 8.28 (m, 1H),9.70 (s, 1H), 12.78 (s, 1H).

EXAMPLE 21

Prepared from the compound 14 and N-methylbutylamine according toGeneral Procedure E. Purification of compound by crystallization inacetic acid gives a gray solid product in 59% of yield, Mp >300° C.(dec.). ¹H-NMR (400 MHz, DMSO-d₆): 12.67 (s, 1H), 9.62 (s, 1H), 8.17 (d,J=2.0 Hz, 1H), 7.90 (m, 2H), 7.80 (d, J=8.8 Hz, 2H), 7.72 (dd, J=1.7 and7.2 Hz, 1H), 7.64 (dd, J=2.1 and 8.6 Hz, 1H), 7.45 (d, J=8.5 Hz, 1H),7.09 (d, J=8.8 Hz, 2H), 5.27 (s, 2H), 4.42 (br s, 2H).

EXAMPLE 22

Prepared from the compound 14 and 3-dimethylphenol according to GeneralProcedure E. Purification of compound by crystallization in acetic acidgives a light yellow solid product in 50% of yield, mp >300° C. (dec.).¹H-NMR (400 MHz, DMSO-d₆): 12.65 (s, br, 1H), 8.16 (d, J=2.0 Hz, 1H),7.91 (m, 2H), 7.72 (dd, J=7.1 and 2.0 Hz, 1H), 7.63 (dd, J=8.6 and 2.1Hz, 1H), 7.44 (d, J=8.5 Hz, 1H), 7.07 (t, J=7.5 Hz, 1H), 6.33 (m, 3H),5.17 (s, 2H), 2.87 (s, 6H).

EXAMPLE 23

Prepared from the compound 14 and 4-(1-imidazole)phenol according toGeneral Procedure E. Purification of compound by crystallization inethanol gives a white solid product in 70% of yield, Mp >300° C. (dec.).¹H-NMR (400 MHz, DMSO-d₆): 12.66 (s, br, 1H), 8.18 (d, J=2 Hz, 1H), 7.91(m, 2H), 7.73)dd, J=7.1 and 1.9 Hz, 1H), 7.66 (dd, J=8.5 and 2.1 Hz,1H), 7.48 (m, 3H), 7.25 (t, J=2.1 Hz, 2H), 7.12 (m, 2H), 6.22 (t, J=2.1Hz, 2H), 5.25 (s, 2H)

EXAMPLE 24

Prepared from the compound 14 and 3-hydroxpyridine according to GeneralProcedure E. Purification of compound by crystallization in ethanolgives a yellow solid product in 57% of yield, Mp >300° C. (dec.). ¹H-NMR(400 MHz, DMSO-d₆): 12.67 (s, br, 1H), 8.39 (d, J=2.9 Hz, 1H), 8.19 (d,J=2.8 Hz, 2H), 7.91 (m, 2H), 7.72 (dd, J=7.1 and 2.0 Hz, 1H), 7.66 (dd,J=8.5 and 2.2 Hz, 1H), 7.49 (m, 2H), 7.35 (m, 1H), 5.30 (s, 2H).

EXAMPLE 25

Prepared from the compound 11 (R=H) and 1.5 equivalent of hydrazine togive benzopyrano[4,3,2-de]phthalazine according to General Procedure C.To a solution of 0.3g of benzopyrano[4,3,2-de]phthalazine in 7.5 mL ofconcentrate sulfuric acid is dropwise added 2.0 mL of fuming sulfuricacid at 0 OC. The mixture is stirred for 3 hours at 0° C. and pouredinto 100 mL of ice-cold water. The precipitation is collected byfiltration, washed with water to give of a white solid product, mp >300°C. (dec.). ¹H-NMR (400 MHz, DMSO-d₆):12.7 (s, 1 H), 8.35 (s, 1 H),7.92-7.90 (m, 2 H), 7.76-7.71 (m, 2 H), 7.38 (d, 1 H).

EXAMPLE 26

Prepared from the compound 16 and N-(2-aminoethyl)morpholine accordingto General Procedure B. Purification of compound by silica gel columnchromatography provides sulfonamide 17. Condensation of compound 17 withhydrazine according to General Procedure C in ethanol affords a solidwhich is purified by crystallization in ethanol gives a white solidproduct in 70% of yield. To this amine compound (10 mmol) in dioxane (10mL) is added 10 mmol of hydrochloric acid in dioxane at 60° C. Whitesalt solid product is formed upon cooling the solution to roomtemperature. The solid is collected by filtration, mp 302-307° C. ¹H-NMR(400 MHz, DMSO-d₆): 12.81 (s, 1H), 10.90 (s, 1H). 8.49 (d, 1H, 2.3 Hz),8.30 (s, 1H), 7.99-7.92 (m, 3H), 7.78-7.75 (dd, 1H, 2.6 Hz, 6.5Hz), 7.65(d, 1H, 8.8 Hz), 3.95-3.92 (m, 2H), 3.78-3.72 (t,2H, 11.8 Hz), 343-3.39(m, 2H), 3.33 (s, 4H), 3.10-3.08 (m, 2H).

EXAMPLE 27

Prepared from the compound 16 and 1-(2-aminoethyl)piperidine accordingto General Procedure B. Purification of compound by silica gel columnchromatography provides sulfonamide 17. Condensation of compound 17 withhydrazine according to General Procedure C in ethanol-water affords asolid which is purified by crystallization in ethanol gives a whitesolid product in 50% of yield. To this amine compound (10 mmol) indioxane (10 mL) is added 11 mmol of hydrochloric acid in dioxane at 60°C. White solid product is formed upon cooling the solution to roomtemperature and collected by filtration. mp >300° C. (dec.). ¹H-NMR (400MHz, DMSO-d₆): 12.82 (s, 1H), 9.80 (s, 1H), 8.49 (d, 1H, 2.4 Hz), 8.26(m, 1H), 7.96-7.94 (m, 3 H), 7.78 (dd, 1H, 2.6 Hz, 6.5 Hz), 7.67 (d, 1H,8.8 Hz), 3.20-3.17 (m, 2H), 3.13-3.12 (m, 2H), 2.89-2.87 (m,2H),1.75-1.68 (m, 6H), 1.45-1.30 (m, 2H).

EXAMPLE 28

Prepared from the compound 16 and N-methylpiperazine according toGeneral Procedure B. Purification of compound by silica gel columnchromatography provides sulfonamide 17. Condensation of compound 17 withhydrazine according to General Procedure C in ethanol affords a solidwhich is purified by crystallization in ethanol gives a white solidproduct in 45% of yield, Mp >300 ° C. (dec.). 1H-NMR (400 MHz, DMSO-d₆):12.82 (s, 1H), 10.55 (s, 1H), 8.36 (d, 1H, 2.3 Hz), 7.96-7.91 (m, 3H),7.79 (dd, 1H, 2.7 Hz, 6.5 Hz), 7.71 (d, 1H, 8.7 Hz), 3.90-3.80 (bs, 2H),3.50-3.40 (bs, 2H), 3.20-3.10 (bs, 2H), 2.85-2.70 (m,5H).

EXAMPLE 29

Prepared from the compound 16 and L-proline according to GeneralProcedure B. Purification of compound by silica gel columnchromatography provides sulfonamide 17. The compound 17 is reacted withhydrazine according to General Procedure C in ethanol affords a solidwhich is purified by crystallization in ethanol gives a white solidproduct in 29% of yield, mp >300 (dec.) ° C. ¹H-NMR (400 MHz, DMSO-d₆):12.78 (s, 1H), 8.41 (d, 1H, J=2.3 Hz), 7.99-7.92 (m, 3H), 7.79 (dd, 1H,J=5.6 Hz, 8.2 Hz), 7.63 (d, 1H, J=8.7 Hz), 4.14 (m, 1H), 3.41-3.32 (m,2H), 1.99-1.90 (m, 1H), 1.90-1.83 (m, 2H), 1.68-1.63 (m, 1H).

EXAMPLE 30

To a solution of compound 14 in m-xylene is added triethyl phosphite themixture is refluxed for 2 hours and cooled to room temperature. Theprecipitation is collected by filtration to give phosphonic ester.Condensation of this compound with hydrazine according to GeneralProcedure C in ethanol affords a solid which is purified bycrystallization in ethanol gives a white solid product in 70% of yield,Mp 196-201° C. LRMS (fab, M+1)=345. ¹H-NMR (400 MHz, DMSO-d₆): 7.36 (t,J=7.8 Hz, 1H), 7.23 (d, J=7.6 Hz, 1H) 7.08 (d, 7.7 Hz, 1H), 6.99 (s,1H), 6.78 (d, J=8.0 Hz, 1H), 6.54 (d, J=8.4 Hz, 1H), 3.53 (d,J=10.4 Hz,3H), 2.82 (d, J=20.1 Hz, 2H).

EXAMPLE 31

Prepared from the compound 11 (R=COOMe) and hydrazine according toGeneral Procedure C. Purification of compound by crystallization inacetonitrile gives a whit solid (40% yield), mp >400° C. (dec.). ¹H-NMR(400 MHz, DMSO-d₆): 12.73 (s, br, 1 H); 9.99 (s, 1H); 8.57 (d, J=2.0 Hz,1H); 7.98 (dd,J=5.5, 2.2 Hz, 1H); 7.92 (m, 2H); 7.33 (dd, J=6.5, 2.6 Hz,1H); 7.47 (d, J=8.7 Hz, 1H); 4.55 (s, 2H).

Other variations and modifications of this invention using the syntheticpathways described above will be obvious to those skilled in the art.

Methods of Using the Compounds of the Invention

The compounds of the present invention can treat or prevent tissuedamage resulting from cell damage or death due to necrosis or apoptosis;can ameliorate neural or cardiovascular tissue damage, including thatfollowing focal ischemia, myocardial infarction, and reperfusion injury;can treat various diseases and conditions caused or exacerbated by PARPactivity; can extend or increase the lifespan or proliferative capacityof cells; can alter the gene expression of senescent cells; and canradiosensitize cells. Generally, inhibition of PARP activity spares thecells from energy loss, preventing, in the case of neural cells,irreversible depolarization of the neurons, and thus, providesneuroprotection. While not being bound to any one particular theory, itis thought that PARP activation may play a common role in still otherexcitotoxic mechanisms, perhaps as yet undiscovered, in addition to theproduction of free radicals and NO.

For the foregoing reasons, the present invention further relates to amethod of administering a therapeutically effective amount of theabove-identified compounds in an amount sufficient to inhibit PARPactivity, to treat or prevent tissue damage resulting from cell damageor death due to necrosis or apoptosis, to effect a neuronal activity notmediated by NMDA toxicity, to effect a neuronal activity mediated byNMDA toxicity, to treat neural tissue damage resulting from ischemia andreperfusion injury, neurological disorders and neurodegenerativediseases; to prevent or treat vascular stroke; to treat or preventcardiovascular disorders; to treat other conditions and/or disorderssuch as age-related muscular degeneration, AIDS and other immunesenescence diseases, arthritis, atherosclerosis, cachexia, cancer,degenerative diseases of skeletal muscle involving replicativesenescence, diabetes, head trauma, immune senescence, inflammatory boweldisorders (such as colitis and Crohn's disease), muscular dystrophy,osteoarthritis, osteoporosis, chronic and/or acute pain (such asneuropathic pain), renal failure, retinal ischemia, septic shock (suchas endotoxic shock), and skin aging; to extend the lifespan andproliferative capacity of cells; to alter gene expression of senescentcells; or to radiosensitize hypoxic tumor cells. The present inventionalso relates to treating diseases and conditions in an animal whichcomprises administering to said animal a therapeutically effectiveamount of the above-identified compounds.

In particular, the present invention relates to a method of treating,preventing or inhibiting a neurological disorder in an animal, whichcomprises administering to said animal a therapeutically effectiveamount of the above-identified compounds. In a particularly preferredembodiment, the neurological disorder is selected from the groupconsisting of peripheral neuropathy caused by physical injury or diseasestate, traumatic brain injury, physical damage to the spinal cord,stroke associated with brain damage, focal ischemia, global ischemia,reperfusion injury, demyelinating disease and neurological disorderrelating to neurodegeneration. Another preferred embodiment is when thereperftision injury is a vascular stroke. Yet another preferredembodiment is when the peripheral neuropathy is caused by Guillain-Barresyndrome. Still another preferred embodiment is when the demyelinatingdisease and neurological disorder relates to neurodegeneration. Anotherpreferred embodiment is when the reperfusion injury is a vascularstroke. Still another preferred embodiment is when the demyelinatingdisease is multiple sclerosis. Another preferred embodiment is when theneurological disorder relating to neurodegeneration is selected from thegroup consisting of Alzheimer's Disease, Parkinson's Disease, andamyotrophic lateral sclerosis.

Yet another preferred embodiment is a method of treating, preventing orinhibiting a cardiovascular disease in an animal, such as anginapectoris, myocardial infarction, cardiovascular ischemia, andcardiovascular tissue damage related to PARP activation, byadministering to said animal an effective amount of the compounds of thepresent invention.

The present invention also contemplates the use of compound I, II, III,IV or V for inhibiting PARP activity, for treating, preventing orinhibiting tissue damage resulting from cell damage or death due tonecrosis or apoptosis, for treating, preventing or inhibiting aneurological disorder in an animal.

In a particularly preferred embodiment, the neurological disorder isselected from the group consisting of peripheral neuropathy caused byphysical injury or disease state, traumatic brain injury, physicaldamage to the spinal cord, stroke associated with brain damage, focalischemia, global ischemia, reperftision injury, demyelinating diseaseand neurological disorder relating to neurodegeneration.

Another preferred embodiment is when the reperfusion injury is avascular stroke. Yet another preferred embodiment is when the peripheralneuropathy is caused by Guillain-Barre syndrome. Still another preferredembodiment is when the demyelinating disease is multiple sclerosis.Another preferred embodiment is when the neurological disorder relatingto neurodegeneration is selected from the group consisting ofAlzheimer's Disease, Parkinson's Disease, and amyotrophic lateralsclerosis.

The present invention also contemplates the use of compound I, II, III,IV or V in the preparation of a medicament for the treatment of any ofthe diseases and disorders in an animal described herein.

In a particular embodiment, the disease or disorder is a neurologicaldisorder.

In a particularly preferred embodiment, the neurological disorder isselected from the group consisting of peripheral neuropathy caused byphysical injury or disease state, traumatic brain injury, physicaldamage to the spinal cord, stroke associated with brain damage, focalischemia, global ischemia, reperfision injury, demyelinating disease andneurological disorder relating to neurodegeneration. Another preferredembodiment is when the reperfusion injury is a vascular stroke. Yetanother preferred embodiment is when the peripheral neuropathy is causedby Guillain-Barre syndrome.

Still another preferred embodiment is when the demyelinating disease ismultiple sclerosis. Another preferred embodiment is when theneurological disorder relating to neurodegeneration is selected from thegroup consisting of Alzheimer's Disease, Parkinson's Disease, andamyotrophic lateral sclerosis.

The term “preventing neurodegeneration” includes the ability to preventneurodegeneration in patients newly diagnosed as having aneurodegenerative disease, or at risk of developing a new degenerativedisease and for preventing further neurodegeneration in patients who arealready suffering from or have symptoms of a neurodegenerative disease.

The term “treatment” as used herein covers any treatment of a diseaseand/or condition in an animal, particularly a human, and includes:

(i) preventing a disease and/or condition from occurring in a subjectwhich may be predisposed to the disease and/or condition but has not yetbeen diagnosed as having it;

(ii) inhibiting the disease and/or condition, i.e., arresting itsdevelopment; or

(iii) relieving the disease and/or condition, i.e., causing regressionof the disease and/or condition.

As used herein, the term “neural tissue damage resulting from ischemiaand reperfision injury” includes neurotoxicity, such as seen in vascularstroke and global and focal ischemia. As used herein, the term“neurodegenerative diseases,” includes Alzheimer's disease, Parkinson'sdisease and Huntington's disease.

The term “ischemia” relates to localized tissue anemia due toobstruction of the inflow of arterial blood. Global ischemia occursunder conditions in which blood flow to the entire brain ceases for aperiod of time, such as may result from cardiac arrest. Focal ischemiaoccurs under conditions in which a portion of the brain is deprived ofits normal blood supply, such as may result from thromboembolyticocclusion of a cerebral vessel, traumatic head injury, edema, and braintumors.

The term “cardiovascular disease” relates to myocardial infarction,angina pectoris, vascular or myocardial ischemia, and related conditionsas would be known by those of skill in the art which involve dysfunctionof or tissue damage to the heart or vasculature, and especially, but notlimited to, tissue damage related to PARP activation.

The term “radiosensitizer”, as used herein, is defined as a molecule,preferably a low molecular weight molecule, administered to animals intherapeutically effective amounts to increase the sensitivity of thecells to be radiosensitized to electromagnetic radiation and/or topromote the treatment of diseases which are treatable withelectromagnetic radiation. Diseases which are treatable withelectromagnetic radiation include neoplastic diseases, benign andmalignant tumors, and cancerous cells. Electromagnetic radiationtreatment of other diseases not listed herein are also contemplated bythe present invention. The terms “electromagnetic radiation” and“radiation” as used herein includes, but is not limited to, radiationhaving the wavelength of 10⁻²⁰ to 10⁰ meters. Preferred embodiments ofthe present invention employ the electromagnetic radiation of:gamma-radiation (10⁻²⁰ to 10⁻¹³ m) x-ray radiation (10⁻¹¹ to 10⁻⁹ m),ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 nm),infrared radiation (700 nm to 1.0 mm), or microwave radiation (1 mm to30 cm).

Compositions and Methods for Effecting Neuronal Activity

Preferably, the compounds of the invention inhibit PARP activity and,thus, are believed to be useful for treating neural tissue damage,particularly damage resulting from cerebral ischemia and reperfusioninjury or neurodegenerative diseases in animals. The term “nervoustissue” refers to the various components that make up the nervous systemincluding, without limitation, neurons, neural support cells, glia,Schwann cells, vasculature contained within and supplying thesestructures, the central nervous system, the brain, the brain stem, thespinal cord, the junction of the central nervous system with theperipheral nervous system, the peripheral nervous system, and alliedstructures.

Further, according to the invention, an effective therapeutic amount ofthe compounds and compositions described above are administered toanimals to effect a neuronal activity, particularly one that is notmediated by NMDA neurotoxicity. Such neuronal activity may consist ofstimulation of damaged neurons, promotion of neuronal regeneration,prevention of neurodegeneration and treatment of a neurologicaldisorder. Accordingly, the present invention further relates to a methodof effecting a neuronal activity in an animal, comprising administeringan effective amount of the compound of formula I, II, III, IV or V tosaid animal.

Examples of neurological disorders that are treatable by the method ofusing the present invention include, without limitation, trigeminalneuralgia; glossopharyngeal neuralgia; Bell's Palsy; myasthenia gravis;muscular dystrophy; amyotrophic lateral sclerosis; progressive muscularatrophy; progressive bulbar inherited muscular atrophy; herniated,ruptured or prolapsed invertebrate disk syndromes; cervical spondylosis;plexus disorders; thoracic outlet destruction syndromes; peripheralneuropathies such as those caused by lead, dapsone, ticks, porphyria, orGuillain-Barré syndrome; Alzheimer's disease; Huntington's Disease andParkinson's disease.

The method of the present invention is particularly useful for treatinga neurological disorder selected from the group consisting of:peripheral neuropathy caused by physical injury or disease state; headtrauma, such as traumatic brain injury; physical damage to the spinalcord; stroke associated with brain damage, such as vascular strokeassociated with hypoxia and brain damage, focal cerebral ischemia,global cerebral ischemia, and cerebral reperfusion injury; demyelinatingdiseases, such as multiple sclerosis; and neurological disorders relatedto neurodegeneration, such as Alzheimer's Disease, Parkinson's Disease,Huntington's Disease and amyotrophic lateral sclerosis (ALS).

Treating Other PARP-Related Disorders

The compounds, compositions and methods of the present invention areparticularly useful for treating or preventing tissue damage resultingfrom cell death or damage due to necrosis or apoptosis.

The compounds, compositions and methods of the invention can also beused to treat a cardiovascular disorder in an animal, by administeringan effective amount of the compound of formula to the animal. As usedherein, the term “cardiovascular disorders” refers to those disordersthat can either cause ischemia or are caused by reperfusion of theheart. Examples include, but are not limited to, coronary arterydisease, angina pectoris, myocardial infarction, cardiovascular tissuedamage caused by cardiac arrest, cardiovascular tissue damage caused bycardiac bypass, cardiogenic shock, and related conditions that would beknown by those of ordinary skill in the art or which involve dysfunctionof or tissue damage to the heart or vasculature, especially, but notlimited to, tissue damage related to PARP activation.

For example, the methods of the invention are believed to be useful fortreating cardiac tissue damage, particularly damage resulting fromcardiac ischemia or caused by reperfusion injury in animals. The methodsof the invention are particularly useful for treating cardiovasculardisorders selected from the group consisting of: coronary arterydisease, such as atherosclerosis; angina pectoris; myocardialinfarction; myocardial ischemia and cardiac arrest; cardiac bypass; andcardiogenic shock. The methods of the invention are particularly helpfulin treating the acute forms of the above cardiovascular disorders.

Further, the methods of the invention can be used to treat tissue damageresulting from cell damage or death due to necrosis or apoptosis, neuraltissue damage resulting from ischemia and reperfusion injury,neurological disorders and neurodegenerative diseases; to prevent ortreat vascular stroke; to treat or prevent cardiovascular disorders; totreat other conditions and/or disorders such as age-related musculardegeneration, AIDS and other immune senescence diseases, arthritis,atherosclerosis, cachexia, cancer, degenerative diseases of skeletalmuscle involving replicative senescence, diabetes, head trauma, immunesenescence, inflammatory bowel disorders (such as colitis and Crohn'sdisease), muscular dystrophy, osteoarthritis, osteoporosis, chronicand/or acute pain (such as neuropathic pain), renal failure, retinalischemia, septic shock (such as endotoxic shock), and skin aging; toextend the lifespan and proliferative capacity of cells; to alter geneexpression of senescent cells; or to radiosensitize tumor cells

Further still, the methods of the invention can be used to treat cancerand to radiosensitize tumor cells. The term “cancer” is interpretedbroadly. The compounds of the present invention can be “anti-canceragents”, which term also encompasses “anti-tumor cell growth agents” and“anti-neoplastic agents”. For example, the methods of the invention areuseful for treating cancers and radiosensitizing tumor cells in cancerssuch as ACTH-producing tumors, acute lymphocytic leukemia, acutenonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer,brain cancer, breast cancer, cervical cancer, chronic lymphocyticleukemia, chronic myelocytic leukemia, colorectal cancer, cutaneousT-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma,gallbladder cancer, hairy cell leukemia, head & neck cancer, Hodgkin'slymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer(small and/or non-small cell), malignant peritoneal effusion, malignantpleural effusion, melanoma, mesothelioma, multiple myeloma,neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer,ovary (germ cell) cancer, prostate cancer, pancreatic cancer, penilecancer, retinoblastoma, skin cancer, soft-tissue sarcoma, squamous cellcarcinomas, stomach cancer, testicular cancer, thyroid cancer,trophoblastic neoplasms, uterine cancer, vaginal cancer, cancer of thevulva and Wilm's tumor.

The term “radiosensitizer”, as used herein, is defined as a molecule,preferably a low molecular weight molecule, administered to animals intherapeutically effective amounts to increase the sensitivity of thecells to be radiosensitized to electromagnetic radiation and/or topromote the treatment of diseases which are treatable withelectromagnetic radiation. Diseases which are treatable withelectromagnetic radiation include neoplastic diseases, benign andmalignant tumors, and cancerous cells. Electromagnetic radiationtreatment of other diseases not listed herein are also contemplated bythe present invention. The terms “electromagnetic radiation” and“radiation” as used herein includes, but is not limited to, radiationhaving the wavelength of 10⁻²⁰ to 10⁰ meters. Preferred embodiments ofthe present invention employ the electromagnetic radiation of:gamma-radiation 10⁻²⁰ to 10⁻¹³ m) x-ray radiation (10⁻¹¹ to 10⁻⁹ m),ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 nm),infrared radiation (700 nm to 1.0 mm), and microwave radiation (1 mm to30 cm).

Radiosensitizers are known to increase the sensitivity of cancerouscells to the toxic effects of electromagnetic radiation. Severalmechanisms for the mode of action of radiosensitizers have beensuggested in the literature including: hypoxic cell radiosensitizers (e.g., 2-nitroimidazole compounds, and benzotriazine dioxide compounds)promote the reoxygenation of hypoxic tissue and/or catalyze thegeneration of damaging oxygen radicals; non-hypoxic cellradiosensitizers (e.g., halogenated pyrimidines) can be analogs of DNAbases and preferentially incorporate into the DNA of cancer cells andthereby promote the radiation-induced breaking of DNA molecules and/orprevent the normal DNA repair mechanisms; and various other potentialmechanisms of action have been hypothesized for radiosensitizers in thetreatment of disease.

Many cancer treatment protocols currently employ radiosensitizersactivated by the electromagnetic radiation of x-rays. Examples of x-rayactivated radiosensitizers include, but are not limited to, thefollowing: metronidazole, misonidazole, desmethylmisonidazole,pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233,EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR),5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine(FudR), hydroxyurea, cisplatin, and therapeutically effective analogsand derivatives of the same.

Photodynamic therapy (PDT) of cancers employs visible light as theradiation activator of the sensitizing agent. Examples of photodynamicradiosensitizers include the following, but are not limited to:hematoporphyrin derivatives, Photofrin, benzoporphyrin derivatives,NPe6, tin etioporphyrin SnET2, pheoborbide-a, bacteriochlorophyll-a,naphthalocyanines, phthalocyanines, zinc phthalocyanine, andtherapeutically effective analogs and derivatives of the same.

Radiosensitizers may be administered in conjunction with atherapeutically effective amount of one or more other compounds,including but not limited to: compounds which promote the incorporationof radiosensitizers to the target cells; compounds which control theflow of therapeutics, nutrients, and/or oxygen to the target cells;chemotherapeutic agents which act on the tumor with or withoutadditional radiation; or other therapeutically effective compounds fortreating cancer or other disease. Examples of additional therapeuticagents that may be used in conjunction with radiosensitizers include,but are not limited to: 5-fluorouracil, leucovorin,5′-amino-5′deoxythymidine, oxygen, carbogen, red cell transfusions,perfluorocarbons (e.g., Fluosol-DA), 2,3-DPG, BW12C, calcium channelblockers, pentoxyfylline, antiangiogenesis compounds, hydralazine, andLBSO. Examples of chemotherapeutic agents that may be used inconjunction with radiosensitizers include, but are not limited to:adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin,docetaxel, doxorubicin, interferon (alpha, beta, gamma), interleukin 2,irinotecan, paclitaxel, topotecan, and therapeutically effective analogsand derivatives of the same.

Pharmaceutical Compositions of the Invention

The present invention also relates to a pharmaceutical compositioncomprising (i) a therapeutically effective amount of the compound offormula I, II, III, IV or V and (ii) a pharmaceutically acceptablecarrier.

The above discussion relating to the preferred embodiments' utility andadministration of the compounds of the present invention also applies tothe pharmaceutical composition of the present invention.

The term “pharmaceutically acceptable carrier” as used herein refers toany carrier, diluent, excipient, suspending agent, lubricating agent,adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent,preservative, surfactant, colorant, flavorant, or sweetener.

For these purposes, the composition of the invention may be administeredorally, parenterally, by inhalation spray, adsorption, absorption,topically, rectally, nasally, bucally, vaginally, intraventricularly,via an implanted reservoir in dosage formulations containingconventional non-toxic pharmaceutically-acceptable carriers, or by anyother convenient dosage form. The term parenteral as used hereinincludes subcutaneous, intravenous, intramuscular, intraperitoneal,intrathecal, intraventricular, intrasternal, and intracranial injectionor infusion techniques.

When administered parenterally, the composition will normally be in aunit dosage, sterile injectable form (solution, suspension or emulsion)which is preferably isotonic with the blood of the recipient with apharmaceutically acceptable carrier. Examples of such sterile injectableforms are sterile injectable aqueous or oleaginous suspensions. Thesesuspensions may be formulated according to techniques known in the artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable forms may also be sterile injectable solutions orsuspensions in non-toxic parenterally-acceptable diluents or solvents,for example, as solutions in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, saline, Ringer'ssolution, dextrose solution, isotonic sodium chloride solution, andHanks' solution. In addition, sterile, fixed oils are conventionallyemployed as solvents or suspending mediums. For this purpose, any blandfixed oil may be employed including synthetic mono- or di-glycerides,corn, cottonseed, peanut, and sesame oil. Fatty acids such as ethyloleate, isopropyl myristate, and oleic acid and its glyceridederivatives, including olive oil and castor oil, especially in theirpolyoxyethylated versions, are useful in the preparation of injectables.These oil solutions or suspensions may also contain long-chain alcoholdiluents or dispersants.

Sterile saline is a preferred carrier, and the compounds are oftensufficiently water soluble to be made up as a solution for allforeseeable needs. The carrier may contain minor amounts of additives,such as substances that enhance solubility, isotonicity, and chemicalstability, e.g., anti-oxidants, buffers and preservatives.

Formulations suitable for nasal or buccal administration (such asself-propelling powder dispensing formulations) may comprise about 0.1%to about 5% w/w, for example 1% w/w of active ingredient. Theformulations for human medical use of the present invention comprise anactive ingredient in association with a pharmaceutically acceptablecarrier therefore and optionally other therapeutic ingredient(s).

When administered orally, the composition will usually be formulatedinto unit dosage forms such as tablets, cachets, powder, granules,beads, chewable lozenges, capsules, liquids, aqueous suspensions orsolutions, or similar dosage forms, using conventional equipment andtechniques known in the art. Such formulations typically include asolid, semisolid, or liquid carrier. Exemplary carriers include lactose,dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calciumphosphate, mineral oil, cocoa butter, oil of theobroma, alginates,tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylene sorbitanmonolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc,magnesium stearate, and the like.

The composition of the invention is preferably administered as a capsuleor tablet containing a single or divided dose of the inhibitor.Preferably, the composition is administered as a sterile solution,suspension, or emulsion, in a single or divided dose. Tablets maycontain carriers such as lactose and corn starch, and/or lubricatingagents such as magnesium stearate. Capsules may contain diluentsincluding lactose and dried corn starch.

A tablet may be made by compressing or molding the active ingredientoptionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing, in a suitable machine, the activeingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, surfaceactive, or dispersing agent. Molded lablets may be made by molding in asuitable machine, a mixture of the powdered active ingredient and asuitable carrier moistened with an inert liquid diluent.

The compounds of this invention may also be administered rectally in theform of suppositories. These compositions can be prepared by mixing thedrug with a suitable non-irritating excipient which is solid at roomtemperature, but liquid at rectal temperature, and, therefore, will meltin the rectum to release the drug. Such materials include cocoa butter,beeswax, and polyethylene glycols.

Compositions and methods of the invention also may utilize controlledrelease technology. Thus, for example, the inventive compounds may beincorporated into a hydrophobic polymer matrix for controlled releaseover a period of days. The composition of the invention may then bemolded into a solid implant, or externally applied patch, suitable forproviding efficacious concentrations of the PARP inhibitors over aprolonged period of time without the need for frequent re-dosing. Suchcontrolled release films are well known to the art. Particularlypreferred are transdermal delivery systems. Other examples of polymerscommonly employed for this purpose that may be used in the presentinvention include nondegradable ethylene-vinyl acetate copolymer andegradable lactic acid-glycolic acid copolymers which may be usedexternally or internally. Certain hydrogels such aspoly(hydroxyethylmethacrylate) or poly(vinylalcohol) also may be useful,but for shorter release cycles than the other polymer release systems,such as those mentioned above.

In a preferred embodiment, the carrier is a solid biodegradable polymeror mixture of biodegradable polymers with appropriate time releasecharacteristics and release kinetics. The composition of the inventionmay then be molded into a solid implant suitable for providingefficacious concentrations of the compounds of the invention over aprolonged period of time without the need for frequent re-dosing. Thecomposition of the present invention can be incorporated into thebiodegradable polymer or polymer mixture in any suitable manner known toone of ordinary skill in the art and may form a homogeneous matrix withthe biodegradable polymer, or may be encapsulated in some way within thepolymer, or may be molded into a solid implant.

In one embodiment, the biodegradable polymer or polymer mixture is usedto form a soft “depot” containing the pharmaceutical composition of thepresent invention that can be administered as a flowable liquid, forexample, by injection, but which remains siifficiently viscous tomaintain the pharmaceutical composition within the localized area aroundthe injection site. The degradation time of the depot so formed can bevaried from several days to a few years, depending upon the polymerselected and its molecular weight. By using a polymer composition ininjectable form, even the need to make an incision may be eliminated. Inany event, a flexible or flowable delivery “depot” will adjust to theshape of the space it occupies with the body with a minimum of trauma tosurrounding tissues. The pharmaceutical composition of the presentinvention is used in amounts that are therapeutically effective, and maydepend upon the desired release profile, the concentration of thepharmaceutical composition required for the sensitizing effect, and thelength of time that the pharmaceutical composition has to be releasedfor treatment.

The PARP inhibitors are used in the composition in amounts that aretherapeutically effective. The compositions may be sterilized and/orcontain adjuvants, such as preserving, stabilizing, welling, oremulsifying agents, solution promoters, salts for regulating the osmoticpressure, and/or buffers. In addition, they may also contain othertherapeutically valuable substances. The compositions are preparedaccording to conventional mixing, granulating, or coating methods, andcontain about 0.1 to 75% by weight, preferably about 1 to 50% by weight,of the active ingredient.

To be effective therapeutically as central nervous system targets, thecompounds of the present invention should readily penetrate theblood-brain barrier when peripherally administered. Compounds whichcannot penetrate the blood-brain barrier can be effectively administeredby an intraventricular route or other appropriate delivery systemsuitable for administration to the brain.

Doses of the compounds preferably include pharmaceutical dosage unitscomprising an efficacious quantity of active compound. By an efficaciousquantity is meant a quantity sufficient to inhibit PARP and derive itsbeneficial effects through administration of one or more of thepharmaceutical dosage units. Preferably, the dose is sufficient toprevent or reduce the effects of vascular stroke or otherneurodegenerative diseases.

For medical use, the amount required of the active ingredient to achievea therapeutic effect will vary with the particular compound, the routeof administration, the mammal under treatment, and the particulardisorder or disease being treated. A suitable systematic dose of acompound of the present invention or a pharmacologically acceptable saltthereof for a mammal suffering from, or likely to suffer from, any ofcondition as described hereinbefore is in the range of about 0.1 mg/kgto about 100 mg/kg of the active ingredient compound, the most preferreddosage being about 1 to about 10 mg/kg.

It is understood, however, that a specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, rate of excretion, drug combination,and the severity of the particular disease being treated and form ofadministration.

It is understood that the ordinarily skilled physician or veterinarianwill readily determine and prescribe the effective amount of thecompound for prophylactic or therapeutic treatment of the condition forwhich treatment is administered. In so proceeding, the physician orveterinarian could employ an intravenous bolus followed by anintravenous infusion and repeated administrations, parenterally ororally, as considered appropriate. While it is possible for an activeingredient to be administered alone, it is preferable to present it as aformulation.

When preparing dosage form incorporating the compositions of theinvention, the compounds may also be blended with conventionalexcipients such as binders, including gelatin, pregelatinized starch,and the like; lubricants, such as hydrogenated vegetable oil, stearicacid, and the like; diluents, such as lactose, mannose, and sucrose;disintegrants, such as carboxymethylcellulose and sodium starchglycolate; suspending agents, such as povidone, polyvinyl alcohol, andthe like; absorbants, such as silicon dioxide; preservatives, such asmethylparaben, propylparaben, and sodium benzoate; surfactants, such assodium lauryl sulfate, polysorbate 80, and the like; colorants such asF.D.& C. dyes and lakes; flavorants; and sweeteners.

The present invention relates to the use of compounds I, II, , III, IVor V in the preparation of a medicament for the treatment of any diseaseor disorder in an animal described herein.

PARP Assays IC₅₀

A convenient method to determine IC₅₀ of a PARP inhibitor compound is aPARP assay using purified recombinant human PARP from Trevigan(Gaithersburg, Md.), as follows: The PARP enzyme assay is set up on icein a volume of 100 microliters consisting of 100 mM Tris-HCl (pH 8.0), 1mM MgCl₂, 28 mM KCl, 28 mM KCl, 28 mM NaCl, 0.1 mg/ml of DNase Iactivated herring sperm DNA (Sigma, Mo.), 3.0 micromolar[3H]nicotinamide adenine dinucleotide (470 mci/mmole), 7 micrograms/mlPARP enzyme, and various concentrations of the compounds to be tested.The reaction is initiated by incubating the mixture at 25° C. After 15minutes of incubation, the reaction is terminated by adding 500microliters of ice cold 20% (w/v) trichloroacetic acid. The precipitateformed is transferred onto a glass fiber filter (Packard Unifilter-GF/B)and washed three times with ethanol. After the filter is dried, theradioactivity is determined by scintillation counting. The compounds ofthis invention were found to have potent enzymatic activity in the rangeof a few nM to 20 μM in IC₅₀ in this inhibition assay.

Using the PARP assay described above, approximate IC₅₀ values wereobtained for the following compounds:

IC₅₀ IC₅₀ Compound (μM) Compound (μM)

0.0131

0.069

0.036

0.052

0.192

0.081

0.208

0.040

0.199

0.198

0.052

0.029

0.390

0.340

0.025

33

0.021

0.045

0.035

0.04

0.025

0.017

0.12

0.072

0.028

2.03

0.128

0.161

0.054

0.044

0.062

0.361

0.017

40

0.089

25

0.139

0.1

0.077

11

0.3610

0.233

0.144

0.058

0.595

0.158

0.059

0.185

0.037

0.085

0.018

0.065

0.023

0.075

Focal Cerebral Ischemia

The following focal cerebral ischemia assay is useful for determiningthe PARP inhibiting effects of the compounds of the present invention.The following examples demonstrate that compunds related to those of thepresent invention are effective in inhibiting PARP activity.

Focal cerebral ischemia is produced by cauterization of the right distalMCA (middle cerebral artery) with bilateral temporary common carotidartery occlusion in male Long-Evans rats for 90 minutes. All proceduresperformed on the animals are approved by the University InstitutionalAnimal Care and Use Committee of the University of Pennsylvania. A totalof 42 rats (weights: 230-340 g) obtained from Charles River were used inthis study. The animals fasted overnight with free access to water priorto the surgical procedure.

Two hours prior to MCA occlusion, varying amounts (control, n=14; 5mg/kg, n=7; 10 mg/kg, n=7; 20 mg/kg, n=7; and 40 mg/kg, n=7) of thecompound, 3,4-dihydro-5-[4-(1-piperidinyl)-butoxy]-1(2H)-isoquinolinone(“DPQ”) were dissolved in dimethyl sulfoxide (DMSO) using a sonicator. Avolume of 1.28 ml/kg of the resulting solution was injectedintraperitoneally into fourteen rats.

The rats were then anesthetized with halothane (4% for induction and0.8%-1.2% for the surgical procedure) in a mixture of 70% nitrous oxideand 30% oxygen. The body temperature was monitored by a rectal probe andmaintained at 37.5±0.50° C. with a heating blanket regulated by ahomeothermic blanket control unit (Harvard Apparatus Limited, Kent,U.K.). A catheter (PE-50) was placed into the tail artery, and arterialpressure was continuously monitored and recorded on a Grass polygraphrecorder (Model 7 D, Grass Instruments, Quincy, Mass.). Samples forblood gas analysis (arterial pH, PaO₂ and PaCO₂) were also taken fromthe tail artery catheter and measured with a blood gas analyzer (ABL 30,Radiometer, Copenhagen, Denmark). Arterial blood samples were obtained30 minutes after MCA occlusion.

The head of the animal was positioned in a stereotaxic frame, and aright parietal incision between the right lateral canthus and theexternal auditory meatus was made. Using a dental drill constantlycooled with saline, a 3 mm burr hole was prepared over the cortexsupplied by the right MCA, 4 mm lateral to the sagittal suture and 5 mmcaudal to the coronal suture. The dura mater and a thin inner bone layerwere kept, care being taken to position the probe over a tissue areadevoid of large blood vessels. The flow probe (tip diameter of 1 mm,fiber separation of 0.25 mm) was lowered to the bottom of the cranialburr hole using a micromanipulator. The probe was held stationary by aprobe holder secured to the skull with dental cement. The microvascularblood flow in the right parietal cortex was continuously monitored witha laser Doppler flowmeter (FloLab, Moor, Devon, U.K., and Periflux 4001,Perimed, Stockholm, Sweden).

Focal cerebral ischemia was produced by cauterization of the distalportion of the right MCA with bilateral temporary common carotid artery(CCA) occlusion by the procedure of Chen et al., “A Model of FocalIschemic Stroke in the Rat: Reproducible Extensive Cortical Infarction”,Stroke 17:738-43 (1986) and/or Liu et al., “PolyethyleneGlycol-conjugated Superoxide Dismutase and Catalase Reduce IschemicBrain Injury”, Am. J. Physiol. 256:H589-93 (1989), both of which arehereby incorporated by reference.

Specifically, bilateral CCA's were isolated, and loops made frompolyethylene (PE-10) catheter were carefully passed around the CCA's forlater remote occlusion. The incision made previously for placement ofthe laser doppler probe was extended to allow observation of the rostralend of the zygomatic arch at the fusion point using a dental drill, andthe dura mater overlying the MCA was cut. The MCA distal to its crossingwith the inferior cerebral vein was lifted by a fine stainless steelhook attached to a micromanipulator and, following bilateral CCAocclusion, the MCA was cauterized with an electrocoagulator. The burrhole was covered with a small piece of Gelform, and the wound wassutured to maintain the brain temperature within the normal ornear-normal range.

After 90 minutes of occlusion, the carotid loops were released, the tailarterial catheter was removed, and all of the wounds were sutured.Gentamicin sulfate (10 mg/mil) was topically applied to the wounds toprevent infection. The anesthetic was discontinued, and the animal wasreturned to his cage after awakening. Water and food were allowed adlibitum.

Two hours after MCA occlusion, the animals were given the same doses ofthe PARP inhibitor as in the pre-treatment. Twenty-four hours after MCAocclusion, the rats were sacrificed with an intraperitoneal injection ofpentobarbital sodium (150 mg/kg). The brain was carefully removed fromthe skull and cooled in ice-cold artificial CSF for five minutes. Thecooled brain was then sectioned in the coronal plane at 2 mm intervalsusing a rodent brain matrix (RBM-4000C, ASI Instruments, Warren, Mich.).The brain slices were incubated in phosphate-buffered saline containing2% 2,3,S-triphenyltetrazolium chloride (TTC) at 37° C. for ten minutes.Color photographs were taken of the posterior surface of the stainedslices and were used to determine the damaged area at eachcross-sectional level using a computer-based image analyzer (NIH Image1.59). To avoid artifacts due to edema, the damaged area was calculatedby subtracting the area of the normal tissue in the hemisphereipsilateral to the stroke from the area of the hemisphere contralateralto the stroke, by the method of Swanson et al., “A Semiautomated Methodfor Measuring Brain Infarct Volume”, J. Cereb. Blood Flow Metabol.10:290-93 (1990), the disclosure of which is hereby incorporated byreference. The total volume of infarction was calculated by summation ofthe damaged volume of the brain slices.

The cauterization of the distal portion of the right MCA with bilateraltemporary CCA occlusion consistently produced a well-recognized corticalinfarct in the right MCA territory of each test animal. There was anapparent uniformity in the distribution of the damaged area as measuredby TTC staining in each group, as shown in FIG. 1.

In FIG. 1, the distribution of the cross-sectional infarct area atrepresentative levels along the rostrocaudal axis was measured from theinteraural line in non-treated animals and in animals treated with 10mg/kg of 3,4-dihydro-5-14-(1-piperidinyl)-butoxy]-1(2H)-isoquinolinone.The area of damage was expressed as mean±standard deviation. Significantdifferences between the 10 mg-treated group and the control group wereindicated (^(*)p<0.02, ^(**)p<0.01, ^(**)p<0.001). The 5 mg/kg and 20mg/kg curves fell approximately halfway between the control and the 10mg/kg curves, whereas the 40 mg/kg curve was close to the control. The5, 20 and 40 mg/kg curves were omitted for clarity.

PARP inhibition led to a significant decrease in the damaged volume inthe 5 mg/kg-treated group (106.7±23.2 mm³, p<0.001), the 10mg/kg-treated group (76.4±16.8 mm³, p<0.001), and the 20 mg/kg-treatedgroup (110.2±42.0 mm³, p<0.01), compared to the control group(165.2±34.0 mm³). The data are expressed as mean±standard deviation. Thesignificance of differences between groups was determined using ananalysis of variance (ANOVA) followed by Student's t-test for individualcomparisons.

There was no significant difference between the control and the 40mg/kg-treated group (135.6±44.8 mm³). However, there were significantdifferences between the 5 mg/kg-treated group and the 10 mg/kg-treatedgroup (p<0.02), and between the 10 mg/kg-treated group and the 40mg/kg-treated group (p<0.01), as shown in FIG. 2.

In FIG. 2, the effect of intraperitoneal administration of3,4-dihydro-5-[4-(1-piperidinyl)-butoxy]-1(2H)-isoquinolinone on theinfarct volume was depicted graphically. The volumes of infarct wereexpressed as mean±standard deviation. Significant differences betweenthe treated groups and the control group were indicated (^(*)p<0.01,^(**)p<0.001). It is not clear why a high dose (40 mg/kg) of the PARPinhibitor, 3,4-dihydro-5-[14-(1-piperidinyl)-butoxy]-1(2H)-isoquinolinone, was lessneuroprotective. The U-shaped dose-response curve may suggest dualeffects of the compound.

However, overall, the in vivo administration of the inhibitor led to asubstantial reduction in infarct volume in the focal cerebral ischemiamodel in the rat. This result indicated that the activation of PARPplays an important role in the pathogenesis of brain damage in cerebralischemia.

The values of arterial blood gases (PaO₂, PaCO₂ and pH) were within thephysiological range in the control and treated groups with nosignificant differences in these parameters among the five groups, asshown below in Table III. A “steady state” MABP was taken followingcompletion of the surgical preparation, just prior to occlusion; an“ischemia” MABP was taken as the average MABP during occlusion.

TABLE III MABP (mm Hg) PaO₂ PaCO₂ Steady Ischemia (mm Hg) (mm Hg) pHState Control 125 ± 21 38.6 ± 4.6 7.33 ± 0.05 79 ± 14 91 ± 13** group (n= 4) 5 mg/kg- 126 ± 20 38.0 ± 2.8 7.36 ± 0.02 78 ± 5  91 ± 12** treatedgroup (n = 7) 10 mg/kg- 125 ± 16 39.3 ± 5.2 7.34 ± 0.03 80 ± 9  90 ±14*  treated group (n = 7) 20 mg/kg- 122 ± 14 41.3 ± 2.8 7.35 ± 0.23 79± 10 91 ± 12** treated group (n = 7) 40 mg/kg- 137 ± 17 39.5 ± 4.7 7.33± 0.24 78 ± 4  88 ± 12*  treated group (n = 7) *= Significantlydifferent from the steady state value, p < 0.05. **= Significantlydifferent from the steady state value, p < 0.01.

There were no significant differences in any physiological parameter,including mean arterial blood pressure (MABP), prior to MCA and CCAocclusion among the five groups Although MABP was significantly elevatedfollowing occlusion in all five groups, there were no significantdifferences in MABP during the occlusion period among the groups.

Since the blood flow values obtained from the laser doppler were inarbitrary units, only percent changes from the baseline (prior toocclusion) were reported. Right MCA and bilateral CCA occlusion produceda significant decrease in relative blood flow in the right parietalcortex to 20.8±7.7% of the baseline in the control group (n=5),18.7±7.4% in the 5 mg/kg-treated group (n=7), 21.4±7.7% in the 10mg/kg-treated group (n=7) and 19.3±11.2% in the 40 mg/kg-treated group(n=7). There were no significant differences in the blood flow responseto occlusion among the four groups. In addition, blood flow showed nosignificant changes throughout the entire occlusion period in any group.

Following release of the carotid occlusions, a good recovery of bloodflow (sometimes hyperemia) was observed in the right MCA territory ofall animals. Reperfusion of the ischemic tissue resulted in theformation of NO and peroxynitrite, in addition to oxygen-derived freeradicals. All of these radicals have been shown to cause DNA strandbreaks and to activate PARP.

This example provided evidence that the related compounds of the presentinvention are effective in inhibiting PARP activity.

Exemplified compounds of the present invention are tested for theirability to reduce focal cerebral ischemia in the following simplifiedprocedure. Rats are allowed free access to water and rat chow (Wayne,Chicago, Ill.) until surgery. Housing and anesthesia concur withguidelines established by the institutional Animal Studies Committee,and are in accordance with the PHS Guide for the Care and Use ofLaboratory Animals, USDA Regulations, and the AVMA Panel on Euthanasiaguidelines.

The animals are anesthetized with isofluorane (induction, 3%;maintenance, 1.25% in a mixture of 30% O₂ and 70% NO₂ through a facemask. The rectal temperature is maintained at 37° C. with a homeothermicblanket (Harvard Apparatus, South Natick, Mass.). First, an iv catheteris inserted into the left femoral vein and the line run up through thenape of the neck for connection to a tethered swivel (InstechLaboratories, Plymouth Meeting, Pa.) and remote infuision pump(Stoelting Inc., Wood Dale, Ill.). In some cases, the right femoralartery is cannulated for monitoring arterial blood pressure and heartrate and for obtaining blood samples for arterial blood gas.

The right middle cerebral artery (MCA) is then exposed by makingvertical skin incision midway between the right eye and ear andoverlying skull is removed with a dental drill (Chen et al, 1986). Afterincision of the dura, the artery is coagulated at the level of theinferior cerebral vein with a bipolar cautery unit (Valleylab NS2000,Boulder, Colo.), and cut to prevent spontaneous reperfusion (Takahashiet al., 1997). Both common carotid arteries (CCAs) that had beenpreviously isolated and freed of soft tissues and nerves are thenligated using non-traumatic aneurysm clips. After the wounds are closedwith surgical clips, the animals are allowed to recover from anesthesiaand returned to their cage which is warmed to 27° C with a heated waterunderpad and humidified warm tent.

The PARP inhibitor to be tested is first administered 30 min after MCAOas an iv bolus, 10 mg/kg infused over 5 min, followed by a 12 hrcontinuous infuision of 2 mg/kg/hr (0.3 ml/hr). Ninety minutes after theMCAO, the animals are removed from the infusion tether, brieflyreanesthetized with isofluorane, and the carotid clips are removed. Theanimal is returned to its warm cage and reconnected to the iv infusionpump for the duration of the experiment.

At 24 hrs after permanent MCAO, animals are sedated with ketamine andthe heads removed by guillotine. Brains are removed, cooled in ice-coldsaline, and sliced into 2 mm coronal sections using a rat brain matrice(Harvard Bioscience, South Natick, Mass.). The brain slices areincubated in phosphate-buffered saline (pH 7.4) containing 2% TTC at 37°C for 10 min. and then stored in 10% neutral-buffered formalin.Cross-sectional area of the TTC-unstained region for each brain slice isdetermined using an image analyzer (MetaMorph, Universal Imaging Corp.,West Chester, Pa.). The total volume of infarction in the righthemisphere is calculated by summation of the direct (TTC-negative) andindirect measures of the infarct areas in the component brain slices.The infarcted volumes in vehicle and drug-treated groups (n=8) aretested for significant statistical difference using an unpairedStudent-t test.

Various doses of the compounds of the invention are tested in thismodel. The compounds are administered in either a single dose or aseries of multiple doses, i.p. or i.v., at different times, both beforeor after the onset of ischemia. Compounds of the invention are expectedto provide protection from ischemia in the range of about 0 to 80%.

Heart Ischemia/Reperfusion Injury

The experiments of the heart ischemia/reperfusion injury model isperformed using female Sprague-Dawley rats weighing 250-300g which areanesthetized with sodium pentobarbital at dose of 65 mg/kgintraperitoneally. The rectal temperature is maintained at 37° C. byusing a homeothermic blanket system (Harvard Apparatus, South Natick,Mass.). The trachea is cannulated and the rat is ventilated with RoomAir by using Harvard Rodent Ventilator (Harvard Apparatus, South Natick,Mass.). The left jugular vein is cannulated with PE-50 tubing for drugdelivery. The right carotid artery is cannulated for BP measurement. Theheart is exposed by opening the chest at the 4th left intercostal space.A main left branch of coronary artery (LAD) is occluded by 4-0 silkligature for 30 min of ischemia and released for 90 min of reperfusion.During the experiment, arterial BP and EKG are monitored by Micro-MedCardiovascular System (Louisville, Ky.).

At the end of reperftision, the LAD coronary artery is re-occluded andabout 2 ml of 5% Evans Blue dye is injected through i.v. line todistinguish the ischemic area from non-ischemic area of the heart. Thenthe heart is immediately taken off and frozen in the freezer. After atleast 30 min of freezing, the heart is sliced into five sections with 2mm thickness and stained in 1% TTC solution for 30 min at 37° C. Theright ventricle is trimmed off. Infarct area, risk area and total leftventricular area in each face of the section are measured by using animage analysis system (BIOQUANT, Nashville, Tenn.). The infarct size iscalculated as the percent total infarct volume of the total risk volume.

For drug treated group, Compounds are administered according to thefollowing three protocols: 1. Single dose of compound is given 60 minprior to the onset of ischemia through i.p. injection. 2. Compound isdelivered through i.v. bolus 1 min before the onset of ischemia followedby i.v. infusion until the end of reperfision. 3. Compound is deliveredthrough i.v. bolus 1 min before the onset of reperfusion followed byi.v. infusion until the end of reperfusion. For each drug-treated group,there is a corresponding vehicle treated group with n=6 or n=8. Thedifference between vehicle and drug treated groups is compared by usingunpaired t-test with p<0.05. Various doses of compounds are tested inthis model. The compounds are given in either single or multiple doses,i.p or i.v., at different times before or after the onset of ischemia.The compounds of this invention are expected to haveischenia/reperfusion injury protection in the range of 10 to 40 percentin this assay.

As a result of the PARP inhibition activity, the compounds of thisinvention are expected to protect against ischemia-induced degenerationof rat cortical neurons in vitro and thus may be useful in disordersarising from cerebral ischemia such as stroke, septic shock, or CNSdegenerative disorders. They may also be useful in protecting the spinalcord following trauma. As an experimental result of ischemia/reperfusioninjury in rats, the present invention is further directed to a method ofprophylactic or therapeutic treatment of heart attack, cardiac arrest,cardiac bypass, diabetes, or risk of damage which comprisesadministering an effective amount of a compound of the present inventionfor PARP inhibition in unit dosage form.

In Vitro Radiosensitization

In vitro radiosensitization may be measured with the use of a humanprostate cancer cell line, PC-3s, which are plated in 6 well dishes andgrown at monolayer cultures in RPMI1640 supplemented with 10% FCS. Thecells are maintained at 37° C. in 5% CO₂ and 95% air. The cells areexposed to a dose response (0.1 mM to 0.1 μM) of 3 different PARPinhibitors prior to irradiation at one sublethal dose level. For alltreatment groups, the six well plates are exposed at room temperature ina Seifert 250 kV/15 mA irradiator with a 0.5 mm Cu/1 mm. Cell viabilityis examined by exclusion of 0.4% trypan blue. Dye exclusion is assessedvisually by microscopy and viable cell number was calculated bysubtracting the number of cells from the viable cell number and dividingby the total number of cells. Cell proliferation rates are calculated bythe amount of ³H-thymnidine incorporation post-irradiation. The PARPinhibitors are expected to radiosensitize the cells.

Measuring Altered Gene Expression in MRNA Senescent Cells

Gene expression alteration may be measured with human fibroblast BJcells which, at Population Doubling (PDL) 94, are plated in regulargrowth medium and then changed to low serum medium to reflectphysiological conditions described in Linskens, et al., Nucleic AcidsRes. 23:16:3244-3251 (1995). A medium of DMEM/199 supplemented with 0.5%bovine calf serum is used. The cells are treated daily for 13 days. Thecontrol cells are treated with and without the solvent used toadminister the PARP inhibitor. The untreated old and young control cellsare tested for comparison. RNA is prepared from the treated and controlcells according to the techniques described in PCT Publication No.96/13610 and Northern blotting is conducted. Probes specific forsenescence-related genes are analyzed, and treated and control cellscompared. In analyzing the results, the lowest level of gene expressionis arbitrarily set at 1 to provide a basis for comparison. Three genesparticularly relevant to age-related changes in the skin are collagen,collagenase and elastin. West, Arch. Derm. 130:87-95 (1994). Elastinexpression of the cells treated with the PARP inhibitor is expected tobe significantly increased in comparison with the control cells. Elastinexpression should be significantly higher in young cells compared tosenescent cells, and thus treatment with the PARP inhibitor should causeelastin expression levels in senescent cells to change to levels similarto those found in much younger cells. Similarly, a beneficial effectshould be seen in collagenase and collagen expression with treatmentwith the PARP inhibitors.

Measuring Altered Gene Expression of Protein in Senescent Cells

Gene expression alteration may be measured with approximately 105 BJcells, at PDL 95-100 which are plated and grown in 15 cm dishes. Thegrowth medium is DMEMW/199 supplemented with 10% bovice calf serum. Thecells are treated daily for 24 hours with the PARP inhibitors of (100μg/1 mL of medium) WO 99/11645. The cells are washed with phosphatebuffered solution (PBS), then permeablized with 4% paraformaldehyde for5 minutes, then washed with PBS, and treated with 100% cold methanol for10 minutes. The methanol is removed and the cells are washed with PBS,and then treated with 10% serum to block nonspecific antibody binding.About I mL of the appropriate commercially available antibody solutions(1:500 dilution. Vector) is added to the cells and the mixture incubatedfor 1 hour. The cells are rinsed and washed three times with PBS. Asecondary antibody, goat anti-mouse IgG (1 mL) with a biotin tag isadded along with 1 mL of a solution containing streptavidin conjugatedto alkaline phosphatase and 1 mL of NBT reagent (Vector). The cells arewashed and changes in gene expression are noted calorimetrically. Foursenescence-specific genes—collagen I, collagen III, collagenase, andinterferon gamma—in senescent cells treated with the PARP inhibitor aremonitored and the results should show a decrease in interferon gammaexpression with no observable change in the expression levels of theother three gens, demonstrating that the PARP inhibitors can altersenescence-specific gene expression.

Extending or Increasing Proliferative Capacity and Lifespan of Cells

To demonstrate the effectiveness of the present method for extending theproliferative capacity and lifespan of cells, human fibroblast cellslines (either W138 at Population Doubling (PDL) 23 or BJ cells at PDL71) are thawed and plated on T75 flasks and allowed to grow in normalmedium (DMEM/M199 plus 10% bovine calf serum) for about a week, at whichtime the cells are confluent, and the cultures are therefor ready to besubdivided. At the time of subdivision, the media is aspirated, and thecells rinsed with phosphate buffer saline (PBS) and then trypsinized.The cells are counted with a Coulter counter and plated at a density of10⁵ cells per cm² in 6-well tissue culture plates in DMEMI99 mediumsupplemented with 10% bovine calf serum and varying amounts (1.0 μM, and1 mM: from a 100× stock solution in DMEM/M199 medium) of a PARPinhibitor. This process is repeated every 7 days until the cells appearto stop dividing. The untreated (control) cells reach senescence andstop dividing after about 40 days in culture. Treatment of cells with 10μM 3-AB appears to have little or no effect in contrast to treatmentwith 100 μM 3-AB which appears lengthen the lifespan of the cells andtreatment with 1 mM 3 -AB which dramatically increases the lifespan andproliferative capacity of the cells. The cells treated with 1 mM 3-ABwill still divide after 60 days in culture.

Neuroprotective Effects of PARP Inhibitors on Chronic ConstrictionIniurv (CCI) in Rats

Adult male Sprague-Dawley rats, 300-350 g, are anesthetized withintraperitoneal 50 mg/kg sodium pentobarbital. Nerve ligation isperformed by exposing one side of the rat's sciatic nerves anddissecting a 5-7 mm-long nerve segment and closing with four looseligatures at a 1.0-1.5-mm, followed by implanting of an intrathecalcatheter and inserting of a gentamicin sulfate-flushed polyethylene(PE-10) tube into the subarachnoid space through an incision at thecisterna magna. The caudal end of the catheter is gently threaded to thelumbar enlargement and the rostral end is secured with dental cement toa screw embedded in the skull and the skin wound is closed with woundclips.

Thermal hyperalgesia to radiant heat is assessed by using apaw-wvithdrawal test. The rat is placed in a plastic cylinder on a 3-mmthick glass plate with a radiant heat source from a projection bulbplaced directly under the plantar surface of the rat's hindpaw. Thepaw-wvithdrawal latency is defined as the time elapsed from the onset ofradiant heat stimulation to withdrawal of the rat's hindpaw.

Mechanical hyperalgesia is assessed by placing the rat in a cage with abottom made of perforated metal sheet with many small square holes.Duration of paw-withdrawal is recorded after pricking the mid-plantarsurface of the rat's hindpaw with the tip of a safety pin insertedthrough the cage bottom.

Mechano-allodynia is assessed by placing a rat in a cage similar to theprevious test, and applying von Frey filaments in ascending order ofbending force ranging from 0.07 to 76 g to the mid-plantar surface ofthe rat's hindpaw. A von Frey filament is applied perpendicular to theskin and depressed slowly until it bends. A threshold force of responseis defined as the first filament in the series to evoke at least oneclear paw-withdrawal out of five applications.

Dark neurons are observed bilaterally within the spinal cord dorsalhorn, particularly in laminae I-II, of rats 8 days after unilateralsciatic nerve ligation as compared with sham operated rats. Variousdoses of PARP inhibitors are tested in this model and shown to reduceboth incidence of dark neurons and neuropathic pain behavior in CCIrats.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications are intended to be included within the scope of thefollowing claims. All references cited herein are incorporated in theirentirety by reference herein.

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